Epigenetic modifications in prostate cancer.

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Epigenetic modifications in prostate cancer

Prostate cancer is the most common cancer in men and the second leading cause of cancer deaths in men in France. Apart from the genetic alterations in prostate cancer, epigenetics modifications are involved in the development and progression of this disease. Epigenetic events are the main cause in gene regulation and the three most epigenetic mechanisms studied include DNA methylation, histone modifications and microRNA expression. In this review, we summarized epigenetic mechanisms in prostate cancer. Epigenetic drugs that inhibit DNA methylation, histone methylation and histone acetylation might be able to reactivate silenced gene expression in prostate cancer. However, further understanding of interactions of these enzymes and their effects on transcription regulation in prostate cancer is needed and has become a priority in biomedical research. In this study, we summed up epigenetic changes with emphasis on pharmacologic epigenetic target agents. Keywords: DNA methylation • epigenetic modifications • histone methylation • microRNA • prostate cancer

Prostate cancer is one of the most prevalent malignancies among men aged 40 to 65. In the carcinogenesis of prostate cancer, somatic epigenetic alterations play a key role. They occur early during the progression of the disease at all stages of the process and are closely related to growth of cancer cells and metastasis progression. There are three major epigenetic changes that include DNA methylation, histone modifications and microRNAs (miRNAs) [1] . It must be understood that hypermethylation of a gene promoter region, histone methylation and acetylation silence gene expression in prostate cancer. But epigenetic aberrations are reversible therefore, modulator enzymes of chromatin that whether demethylate DNA or inhibit histone methylation as well as HDACs can restore tumor suppressor gene function in prostate cancer. Notably, genes silenced by epigenetic alterations are defined as potential biomarkers in prostate cancer diagnosis and can be used to develop effective drug therapies for prostate cancer. As it can be seen, this

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study summarizes epigenetic and regulatory changes that are involved in prostate cancer. DNA methylation & prostate cancer DNA methylation is one of the best wellknown epigenetic modifications that have been widely studied in prostate cancer. New technologies have contributed to a better understanding of the underlying molecular mechanisms of prostate cancer. DNA methylation of CpG islands is associated with inheritable silencing of gene expression and is responsible for physiologic processes such as silencing of repetitive and centromeric sequences, cell differentiation during development, X-chromosome inactivation, genomic imprinting and susceptibility to genetic diseases [2] . Additionally, DNA methylation is catalyzed by enzymes called DNMTs. There are two main DNMT groups, including maintenance of methylation via DNMT1 and de novo methylation via DNMT3A and DNMT3B [3] . Studies reported that DNMT1 activity is related

Epigenomics (2014) 6(4), 415–426

Marjolaine Ngollo1,2, Aslihan Dagdemir1,2, Seher KarsliCeppioglu1,3, Gaelle Judes1,2, Amaury Pajon1,2, Frederique Penault-Llorca1,2, JeanPaul Boiteux4, Yves-Jean Bignon*,1,2, Laurent Guy2,4 & Dominique J BernardGallon1,2 Department of Oncogenetics, Centre Jean Perrin, CBRV, 28 place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France 2 EA 4677 ERTICa, University of Auvergne, 28 place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France 3 Department of Toxicology, Faculty of Pharmacy, Marmara University of Istanbul, Turkey 4 Department of Urology, Gabriel Montpied Hospital, 58 rue Montalembert, 63000 Clermont-Ferrand, France *Author for correspondence: yves-jean.bignon@ cjp.fr 1

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Review Ngollo, Dagdemir, Karsli-Ceppioglu et al. to the transition from non-neoplastic to neoplastic phenotype whereas the de novo methylation enzymes were mainly related to progression. In any case, levels of DNMT1, DNMT3a and DNMT3b were higher in prostate cancer compared with benign prostatic hyperplasia tissue samples, and significantly higher in cultures derived from prostate cancer correlating with high Gleason score linked to poor prognosis [4] . Abnormal DNA methylation in specific regions generates genomic disorders. Indeed, loss of methylation of CpG dinucleotides that are normally methylated can lead to oncogenes activation and therefore, genomic instability. Clearly, cancer initiation and progression determine the changes in DNA methylation [5] . For the most part, two major types of altered DNA methylation have been observed in human cancers, including hypomethylation and hypermethylation of CpG islands. Hypomethylation usually involves repeated DNA sequences whereas hypermethylation involves CpG islands. Loss and/or gain of methylation in specific regions contribute to carcinogenesis [6] . Undoubtedly, DNA hypomethylation plays a role in carcinogenesis because it causes aberrant gene expression, microsatellite instability, loss of imprinting, chromosomal instability and abnormality and activation of retrotransposons [7] . Recent study showed that some oncogenes are hypomethylated and up-regulated in prostate cancer. It is the case of NCOA4 that interacts with the androgen receptor in a ligand-dependent manner to enhance its transcriptional activity. Also, FGF6, a member of the fibroblast growth factor group, which possesses broad mitogenic and cell survival activities, is involved in a variety of biological processes. Furthermore, FGF6 plays an essential role in embryonic development, cell growth, morphogenesis, tissue repair and tumor growth and invasion [8] . Another key point is that, the DNA hypermethylation of oncosuppressor promoters causes the inhibition of their transcripts and contributes to prostate carcinogenesis. In effect, this cytosine methylation in the promoter regions prevents binding of transcription factors and consequently, the transcription of target genes. However, these mechanisms are regulated and reversible [9–11] . Many of the hypermethylated genes in prostate cancer are tumor suppressor genes that coded for the proteins that regulate the cell cycle and/or promote apoptosis. Dysfunction of these genes by promoter hypermethylation can contribute to the initiation and progression of prostate cancer. Actually, the most studied genes are the ones involved in DNA repair for instance, the GSTP1, which is the most frequently downregulated gene by hypermethylation in prostate cancer making it an excellent diagnostic marker [12] . The another one, the MGMT is an epigenetic marker of early prostate tumor

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development [13] . Other genes involved in cell cycle control are CDKN2A that inhibit the cell cycle via the cyclin-dependent kinase [14] . The most compelling evidence is that methylation of other genes such as RASSF1, is strongly correlated with an increased risk of prostate cancer recurrence, aggressiveness and tumor progression [15] . Surely, RASSF1 interacts with the kinase AURORA E-CADHERIN involved in epithelial-to-mesenchymal transition. The rate of methylation of these genes in prostate cells differs from one study to another, depending on the technique and/or biological material used. For sure, higher methylation of certain genes such as APC has been studied in vivo and in vitro in prostate cancer. In fact, these genes are potential biomarkers in prostate cancer. Up to now, more than 50 genes have been proved to be inactivated by promoter hypermethylation in prostate cancer [16] . Hypermethylation, can also hinder transcription at the promoter regions of tumorsuppressor genes, including p16INK4a, P53, RB1 and BRCA1 [17] . Some specific genes implicated within each pathway category are summarized in Table 1. Important to realize is the fact that full understanding of hypermethylation of tumor suppressor genes and DNMTs overexpression in prostate cancer helped identify the biomarkers and develop demethylating agents such as promising anticancer drugs. As a result, in recent years, several DNA methyltransferase inhibitors have been developed. Obviously, demethylating agents can restore both the normal transcription process and the normal function of silenced process genes. On that account, DNA methylation inhibitors might constitute an alternative therapy in prostate cancer [18] . Although, recent advances in diagnostic tools have significantly increased early detection of prostate cancer biomarkers, the challenge remains in the possibility to individualize the diagnosis and treatment of prostate cancer. But, the discovery of next-generation sequencing technologies such as methylated DNA immunoprecipitation sequencing and reduced representation bisulfite sequencing will allow the extension of the prostate cancer biomarkers list [19,20] . In order to map up wholegenome CpG dinucleotide methylation, studies have used whole-genome methylation sequencing method. This study showed that in benign prostate that are adjacent to tumor tissue, the epigenetic alterations, precisely promoter methylation are quite similar to those in prostate cancer, suggesting a critical DNA methylation role in prostate cancer. [8] . Moreover, Gao et al. (2014) by using the ChIP-BS-Seq method demonstrated that a set of genes, commonly enriched with H3K27me3 marks in cancer cells, presented impaired methylation of promoters [21] . In spite of the complexity of the link between histone modification and DNA methylation, the H3K27me3-bound hypermethylated promoter

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in specific genes might be considered as potential new prognostic biomarkers and targeted therapeutic agents. Histone modifications & prostate cancer Histone modifications play a major role in the development and progression of prostate cancer. N-terminal tails of histones are subject to post-translational modifications, including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP ribosylation and deamination. All these modifications constitute the histone code [33] . In the subsequent step of this review, more attention will be paid on histone methylation and acetylation and of course, their role in prostatic carcinogenesis. Simply, histone modifications can be analyzed by chromatin immunoprecipitation (ChIP) method. This technique allows the analysis of specific genomic regions that are associated with protein, such as transcription factors or histone marks [34] . Recently, next generation of DNA sequencers have allowed to determine the wide genome distribution of specific modification and measure the fold enrichment of each locus by ChIP-sequencing and ChIP-on-chip method. Histone methylation/demethylation

DNA methylation is not sufficient to explain silencing of genes in prostate cancer. Other mechanisms such as histone modifications also contribute to gene silencing.

Review

One of these mechanisms is histone methylation that can occur on two different residues, the lysine and arginine with in effect on chromatin structure and gene expression. Histone methylation can lead to activation or inhibition of gene expression, depending on the target amino acid residues and the range of methylation (me1, me2 or me3). Generally, H3K4, H3K36 and H3K79 methylations are associated with active genes while H3K9, H3K27 and H4K20 methylations are considered as repressive marks leading to inactive genes. Again, histone methylation is catalyzed by enzymes called HMTs while the reverse reaction is catalyzed by HDMs. These histone methylation modifying enzymes play a considerable role in prostate cancer progression. Unfortunately, DNA methylation and histone methylation are studied to a lesser extent in prostate cancer. All methyltransferase enzymes contain a conserved methyltransferase SET domain , which is crucial for catalytic activity [35] . Histone methyltransferase enzymes have been associated with prostate cancer development. For example, it was demonstrated that EZH2, part of a polycomb repressive complex 2 (PRC2) plays a major role in prostate cancer development and controls transcriptional repression through different mechanisms [36] . In this section, because EZH2 plays a key role in the development of the disease, the study focuses only on the EZH2 protein.

Table 1. Summary of some specific hypermethylated genes implicated in prostate cancer. Gene name

Functions

GSTP1

Plays a crucial function in the detoxification of both endogenous and exogenous carcinogens

[22]

RASSF1A

Tumor suppressor gene, cell cycle regulator that controls transition from G1 to S phase

[23]

APC

Tumor suppressor gene that regulates the Wnt signaling pathway via ubiquitin-mediated β-catenin degradation

[12]

MGMT

DNA repair protein responsible for the cellular defense against DNA alkylation damage in human cells

[24]

CCNA1

Cell cycle regulator factor involves G1/S cell cycle progression and G2/M phase transition

[25]

CDH1

Suppressor tumor gene involves invasion and proliferation

[26]

RUNX3

Plays a critical role in the regulation of cell proliferation, cell death by apoptosis and cell adhesion and functions as a tumor suppressor gene

[27]

ESR2

Nuclear receptor transcription factor plays a role in differentiation and development of prostate

[28]

CDKN2A

Plays a major role in cell cycle regulation. Acts as a tumor suppressor

[29]

RARβ2

Tumor suppressor gene in human prostate cancer cells. Regulates gene expression in various biological processes

[30]

IGFBP 7

Regulates insulin pathway

[31]

SFRP2

Acts as soluble modulators of Wnt signaling

[32]


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Review Ngollo, Dagdemir, Karsli-Ceppioglu et al. For optimal methyltransferase activity, EZH2 interacts with SUZ12, EED and RbAp48 [37] . Studies suggested that there are additional PRC2 components,AEBP2 and JARID2 but are destitute of histone demethylase activity [38] . These proteins act as accessory units in regulating the function and enzymatic activity of PRC2. EZH2 is also known to interact with other epigenetic machineries such as HDACs and DNMTs, predisposing DNA to methylation and strengthening the epigenetic repressed target genes [39] . In prostate cancer, EZH2 overexpression is linked to an increase of H3K27me3 marks with the consequence on downregulation of several genes leading to amplification of tumor cell proliferation [40] . Recently, it was also showed that H3K27me3 marks were significantly higher in peri-tumoral tissues of prostate cancer patients compared with normal tissue. In reality, these results suggest that alterations in histone methylation occur in peri-tumoral tissues (tissues adjacent to tumor) and that these alterations are quite similar to those in prostate cancer and established H3K27me3 as an epigenetic mark pathogenically involved in neoplasia trough the silencing of genes [41] . Likewise, EZH2 is involved in the regulation of cycle cell progression and proliferation. In fact, EZH2 is regulated by E2F1 that regulate the transition from G2-M phase of the cell cycle [42] . Also, it has been previously shown that p53 is involved in EZH2 regulation, repressing transcription to maintain genetic stability. Therefore, in prostate cancer the loss of p53 expression is associated with an EZH2 increased activity [43] . Furthermore, DNA methylation and histone modifications are closely related through methyl CpG binding proteins, which act as adapters between methylated DNA and chromatin modifying factors. Practically, methyl CpG binding proteins recruit co-repressors such as HDACs, HMTs or chromatin remodeling factors to establish the complex which leads to transcription repression [44] . New powerful technologies using widescale molecular applications, such as ChIP-sequencing or ChIP-chip have permitted to study the relationship between histone modifications and gene transcription in prostate cancer, and as well as identified new molecular mechanisms which are accountable for cancer initiation and progression. Previous studies had worked on genomewide profiling of H3K4me3 H3K27me3 using ChIPchip techniques in prostate cancer cells. As a result, they demonstrated that H3K27me3 marks were higher than H3K4me3 marks in prostate cancer cells, while the result is different from normal prostate cells. Basically, these results suggest that the polycomb system is globally more active in prostate cancer cells than in noncancerous ones. This indicates that H3K4me3/H3K27me3 is an epigenetic signature of prostate carcinogenesis

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[45,46] .

In addition, global ChIP-sequencing analysis has revealed EZH2 binding and a significant enrichment of repressive histone mark H3K27me3 around AR-repressed genes, which suggested that EZH2 was involved in AR-repressed gene expression [47] Since the discovery of HDM molecules, histone methylation was considered as an irreversible epigenetic mark. The new class of histone lysine demethylases (KDMs) enzymes can remove both repressive and active histone marks. Several enzymes that print or read histone methylation marks have been shown to be affected in prostate cancer. Histone methylation is removed by a wide variety of specific enzymes of lysine residues (Table 2). Thereby, KDMs could represent diagnostic tools in obtaining new therapeutic agents against prostate cancer. The first identified histone demethylase is LSD1 also known as KDM1A, which demethylates H3K9me1 or me2 and H3K9 me1 or me2. However, LSD1 protein acts both as a co-activator and a transcriptional co-repressor. Indeed, demethylation of H3K4 is dependent on the other post-translational modifications of histone H3 and the presence of cofactors. When LSD1 is associated with corepressor, CoREST, LSD1 demethylates H3K4. Nevertheless, when it recruited by androgen receptor, LSD1 catalyzes the H3K9 demethylation [48,49] . Another demethylase involved in prostate cancer is JMJD2C also known as KDM4C, member of the Jumonji domain 2 (JMJD2) class. This nuclear protein functions as a trimethylation-specific demethylase and is the first histone tri-demethylase regulating androgen receptor function. Also, JMJD2C interacts with AR in vitro and in vivo. Definitely, the fixation of JMJD2C and AR on androgen receptor-target genes results in demethylation of H3K9me3 and stimulation of androgen receptor-dependent transcription [50] . H3K27me3 demethylation is regulated by UTX/ JMJD3 proteins. In fact, JMJD3 causes a specific reduction of H3K27me3, but had no effect on H3K27me2 or H3K27me1. JMJD3 has been shown to be up-regulated in prostate cancer [51] . To emphasize, previous studies have reported that JMJD3 expression is limited in benign prostate, but is up-regulated in prostate cancer and its expression is even higher in metastatic disease, suggesting that the expression levels of JMJD3 increase with disease severity [52] . The following demethylase (JMJD2A, JMJD2B, JMJD2C, JMJD1A and LSD1) are often overexpressed in prostate cancers and promote aberrant activation of AR target genes. Among the 30 JmjC proteins in the human genome, 18 possess histone demethylase activity. In this case, JmjC proteins can be considered as the main recruiter of diverse genomic loci. For that reason, their impact on specific gene expression is considerably insignificant [53] .



Review

Table 2. Summary of some histone demethylase enzymes. Histone modifying enzyme

Target modification Cellular function

Ref.

LSD1/KDM1

H3K4me1/me2, H3K9me1/me2

Transcriptional repression

[55]

KDM4A/JMJD2A

H3K9me2/3, H3K36me2/me3

Transcriptional activation

[56]

KDM4C/ JMJD2C

H3K9me2/me3, H3K36me2/me3


[50]

KDM6B/JMJD3

H3K27me2/me3


[51]

KDM6A/UTX

H3K27me2/me3


[52]

JMJD : Jumonji domain 2; KDM : Lysine (K)-specific demethylase; LSD1: Lysine-specific demethylase1.

Clearly, prostate cancer is thought to occur initially as an androgen-dependent tumor that, in some cases, can progress to a highly invasive androgenindependent tumor. Some KDM enzymes have already been reported as co-activators of the AR, including KDM4D, KDM4A, KDM1A and KDM4B [54] . Histone acetylation/deacetylation

Histones are basic proteins that increase the affinity of negatively charged DNA due to their positively charged residues. They are acetylated on multiple lysine residues, with the most well-studied acetylations being those that occur on the N-terminal tails of histones, particulary on H3K9, H3K14, H4K16, H4K8 and H4K12 [57] . In principle, acetylation of lysine residues on histone H3 and H4 leads to chromatin structure, resulting in increased transcription except, H3K4me3 which clearly matches with actively transcribed genes. Studies have demonstrated that H3K4me3 is located around the transcription initiation sites and enhanced the transcribed genes. Set1/COMPASS family proteins including, MLL1-4 were the first identified H3K4 methylase and are capable of catalyzing the H3K4me1, me2 and me3 [58] . The mixed lineage leukemia (MLL) acts in opposition to PRC2 activating gene expression. Previous study has reported that UTX was co-purified with MLL3/MLL4 complexes [59] . Along with UTX, MLL3/4 activity represents a distinct form of antagonizing Polycomb function. These two genes are frequently mutated in human cancer causing silencing of genes. Two major types of HATs have been identified: type A, the most nuclear and type B the most cytoplasmic. The type B HATs are involved in acetylation of newly synthesized histones prior to their import into the nucleus. However, the type A HATs are relatively multiform and can be divided into five groups: GNATs, MYST, P300/CREB, general transcription factor and nuclear hormone-related HATs. These enzymes modify multiple sites within the histone N-terminal tails. When these complexes are associated with the histones,


they bridge the gaps and bring close transcription factors, which can be far away and the promoters by acetylating histones. They induce a loosening of chromatin to allow binding of transcription factors that directly interact with DNA promoters [60] . Several type A HATs are involved in prostate cancer, including p300, PCAF and Tip60. These HAT enzymes make the chromatin environment more easily accessible to the transcriptional machinery, increasing transcriptional activity of many genes [61] . As a matter of fact, AR signaling is crucial in the initiation and progression of prostate cancer. Transcriptional activity of AR involves chromatin recruiting co-activators, such as P300, PCAF and SRC3 through histone modifications. On the positive side, it was recently shown that the inhibition of p300 slows down proliferation, stimulates apoptosis and inhibits migration and invasion [62] .The PCAF has been shown to act as a co-activator to regulate the AR transcription and it up-regulates itself in prostate cancer . Up-regulation of PCAF promotes AR transcriptional activation and cell growth in prostate cancer cells. However, another study showed that miR-17–5p targets PCAF mRNA translation and induces its degradation, suggesting that up-regulation of PCAF in prostate cancer may be associated with downregulation of miR-17-5p [63] . Tip60 HAT enzyme is involved in multiple cellular processes, including transcriptional regulation, DNA damage repair and cell signaling. In prostate cancer, several cases overexpress Tip60 which functions as an androgen receptor co-activator [54] . The HDACs are a group of enzymes that are responsible for the deacetylation of lysine residues at the N-terminal regions of core histones. Many of these HDAC substrates are the regulatory proteins that are involved in cell adhesion, cell division and apoptosis. HDACs regulate gene expression and other cellular functions by deacetylating the lysine residues in the proteins substrate. For example, histones are among the most promising targets for anticancer drug development.


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Review Ngollo, Dagdemir, Karsli-Ceppioglu et al. It has been demonstrated that HDAC1 and HDCA3 are overexpressed in prostate cancer, suggesting that they play roles in the inactivation of various genes [64] . Also, HDACs reduce histone acetylation leading to a decrease of H3K4 methylation and increase of H3K9 methylation, playing then a critical role in the maintenance of DNA promoter methylation- associated RASSF1A gene silencing [65] . MiRNA(s) & prostate cancer The last 10 years have demonstrated remarkable advances in the understanding of miRNA biology. MiRNAs are defined as small noncoding RNA molecules that regulate the expression of multiples genes by deteriorating mRNAs stability and/or interrupting translation. MicroRNAs play an important role in the control of cell proliferation and tumorigenesis through gene regulation. The MicroRNA expression becomes altered during the development and progression of prostate cancer. During the process of tumorigenesis, changes are observed in microRNA expression that are characterized by a loss of oncosuppressor microRNAs and overexpression of oncomiRNAs [66] . Also, the deregulation of miRNAs occurs during prostate cancer and altered miRNA expression. It may serve as a biomarker for prostate cancer diagnosis and treatment. Previous study on promoters of human miRNA using ChIP-chip analysis have shown that 28 miRNAs were up-regulated and 30 downregulated in prostate cancer cells compared with normal cells. Among these miRNAs, some are more applicable to prostate cancer, such as miR-205 and miR-200b [67] . Other recent studies report that androgen represses the miR-99a/let7c/125b-2 cluster through AR and anti-androgen drugs block the androgen-repression of the miRNA cluster, suggesting that downregulation of the miR-99a/let7c/125b-2 cluster by androgen protects many of their target mRNAs from degrading and indirectly assists the gene induction [68] . Subsequent study has shown a relationship between miRNA and EZH2 regulation in prostate cancer. In normal cells, miR-101 is expressed and bound to target sites in the 3′ untranslated region of the EZH2 mRNA via the RNA-induced silencing complex, leading to translational repression and/or mRNA destabilization. On the contrary, in cancer cells, miR-101 expression is decreased leading to increased EZH2 translation, altered tumor suppressor and pro-differentiation gene silencing via H3K27me3 repressive histone modifications [69] . According to a recent study on prostate cancer tissues using microarray method, 82 differentially expressed miRNA have been revealed in high-risk prostate cancer. Based on these results, the authors selected seven miRNAs that are found to be differ-

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ently expressed in prostate cancer tissue. Among the selected miRNAs, three belong to the let-7 group and were progressively downregulated in patients with poor cancer indication characterized by a high Gleason Score [70] . DNA hypermethylation of CpG sites within CpG islands is also known to lead to the inactivation of many tumor suppressor miRNAs. For example, recent study has reported that miR-132 is significantly downregulated through CpG hypermethylation in prostate cancer and correlated with clinicopathological characteristics of patients, suggesting that miR-132 might have a prognostic value in prostate cancer [71] . Also, miR-199a-3p is downregulated in prostate cancer tissue and expression level of miR-199a-3p is inversely correlated with tumor stage and Gleason score of prostate cancer [72] . In the same way, the same study has also shown that miR-34b acts as a tumor suppressor in prostate cancer. Also, it showed that miR-34b is silenced through DNA hypermethylation leading to low expression. MiR-34b has antiproliferative and antimigratory effects partly by inhibiting the AKT pathway and mesenchymal markers. Indeed, miR-34b suppressed phosphorylation of AKT (Ser473) and its downstream target GSK3 (Ser9), which in turn becomes activated. There is a series of events that starts with the activation of GSK3 which afterward phosphorylates the β-catenin leading to its degradation and finally to a decreased expression of proliferative genes, such as c-myc and cyclin D1 [73] . Equally important, another recent study has shown that miR-494–3p has anti-tumoral effect in prostate cancer and might suppress progression and metastasis. MiR-494–3p is a post-transcriptional regulator of CXCR4. This gene encodes a CXC chemokine receptor specific to stromal cell-derived factor-1 and is highly expressed in prostate cancer cells. These results suggested that miR-494–3p/CXCR4 pathway may be a potential therapeutic target to prevent prostate cancer progression and metastasis [74] . Other miRNAs were identified as a new prostate cancer miRNA biomarker by using bioinformatics framework such as miR-648, miR-155 [75] , miR-103, miR-125b and miR-222 [76] . It is known that HDACs are up-regulated in prostate cancer even though previous report showed that HDAC1 is a direct target of miR-449a. In fact, a decrease of miR-449a expression results in HDAC1 overexpression. Certainly, miR-449a plays an important role as a tumor suppressor because it stops cellular growth in prostate cancer [77] . In the final analysis, microRNAs are important regulators of gene expression. They can therefore serve as prognostic marker to provide a great tool in the prostate cancer treatment.



Pharmacological targets Some therapies have been developed targeting the epigenetic changes in prostate cancer. In this section, our focus will be on DNA methylation inhibitors, histone deacetylase inhibitors and histone methyltransferase inhibitors. Some epigenetic drugs used in prostate cancer cell lines are summarized in Table 3. DNMT inhibitors

Actually, 5-aza-cytidine has been shown to inhibit DNMT activity once incorporated into DNA. The 5-aza-cytidine is the most investigated and well-known DNA demethylation agent, the first DNA methyltransferase inhibitor, which removes methyl residues from silenced genes, generating re-expression of these genes in many types of cancers, including prostate cancer. Another nucleoside analog that inhibits DNMT is 5-aza2’-deoxycitidine. Some studies revealed that these two molecules may significantly reactivate dormant genes in prostate cancer such as P16, CDKN1C and RASSF1 [78] . Recently, much attention has been paid on the small non-nucleoside DNMT inhibitor. For example the RG108, that directly binds to the catalytic region of DNMT leading to significant inhibition and apoptosis induction in prostate cancer cells. The RG108 also displays a DNMT decreased activity. Interestingly, treatment based on RG108 significantly decreases GSTP1, APC and RARβ2 promoter hypermethylation levels. This occurrence is likely mediated by reversion of aberrant DNA methylation that affects cancer-related genes, which are epigenetically silenced in prostate cancer [79] . However, the most efficient demethylating agents remain by far the DNMT antisense or siRNA that react against DNMT. Similarly, natural molecules also have demonstrated their protective effects against prostate carcinogenesis

Review

or its progression. The two major natural molecules in the row include the genistein and daidzein,, which are the phytoestrogens of soy that have been reported to have the ability to reverse DNA hypermethylation in cancer cell lines on BRCA1, GSTP1, EPHB2 and BRCA2 promoter genes [9,11] . Likewise, a recent study has demonstrated that daidzein and genistein showed a synergistic effect on the cell proliferation inhibition and apoptosis induction in prostate cancer cells [80] . HDAC inhibitors

It must be understood that the mechanism that leads to cancer initiation and promotion is partly due to epigenetic regulation through impaired activation of HDACs. An emphasis has been put on the epigenetic therapy search that governed to the discovery of many encouraging anti-neoplastic agents, especially the HDAC inhibitors successfully used in clinical trials. The HDAC inhibitors are a new class of anticancer agents that act by inhibiting cancer cell proliferation and inducing apoptosis in various cancer cell lines. Among these HDAC inhibitors, the following have been specifically studied in prostate cancer cell lines, they consist of trichostatin A, depsipeptide, MS-275, sodium butyrate (NaBu), valproic acid, pyroxamide and suberoylanilide hydroxamic acid (SAHA) [81] . Particularly, SAHA has proven remarkable effects on the caspase-dependent apoptosis associated with chromatin condensation, DNA fragmentation and mitochondrial membrane depolarization. Another HDAC inhibitor, the NaBu is the short chain fatty acid that plays an anticancer role without toxic effect on normal cells. It therefore would be considered as a potential molecule for anticancer therapeutics in prostate cancer. Another piece of work showed that NaBu treatment modifies histone acetylation,

Table 3. Examples of epigenetics drugs tested in prostate cancer cell lines. Names

Chemical structures

Targets

5-aza-cytidine

Nucleoside analog

DNMT1

5-aza-2’-deoxycytidine

Nucleoside analog

DNMT1

Vorinostat

Hydroxamic acid

HDACs

MHY219

Benzamide

HDACs I and II

NaBu

Fatty acid

HDAC I

Romidepsin

Cyclic peptide

HDACs I and II

Nucleoside

EZH2

DNMT inhibitors

HDAC inhibitors

HMT inhibitor 3-deazanoplanocin A

DNMT: DNA methyltransferase; EZH2: Enhancer of zeste homolog 2; HDAC: Histone deacetylase; HMT: Histone methyltransferase; NaBu: Sodium butyrate.



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Review Ngollo, Dagdemir, Karsli-Ceppioglu et al. affects expressions of co-repressor (SMRT) and coactivators (P300) of AR, and accordingly affects the transcriptional activity of AR. Unfortunately, P300 promotes the progression of prostate cancer by activating the AR and consequently the regulation of androgen-dependent genes. However, in the prostate cancer treatment, the NaBu increases SMRT expression level while the P300 expression level is decreased. By increasing the SMRT expression level, the NaBu contributes to lowering the AR transcription. Furthermore, NaBu increases acetylation of lysine 8 and 12, part of histone H4 in prostate cancer cells compared with normal cells [82] . Latterly, a new molecule called MHY219 has been discovered. This molecule increases histone H3 acetylation and reduces the expression of all HDAC1 and HDAC2 classes in prostate cancer cells. Besides, MHY219 acts at the cellular cycle level by increasing the sub-G1 fraction of cells through p21- and p27-dependent pathways in prostate cancer cell lines (DU145). Also, MHY219 significantly induces the blockage of the G2/M phase in prostate cancer cell lines (DU145 and PC3) and stops the cell cycle at G0/G1 phase in LNCaP cell line. Additionally, MHY219 induces apoptosis in prostate cancer cells through an increase of the cleavage of mitochondrial proteins, PARP and Bax, plus cytochrom C release and Bcl-2 expression decrease. Some authors demonstrated that MHY219 is more effective than the SAHA in treating human prostate cancer cells [83] . Basically, these molecules that target multiple aspects of regulation of cancer cells death might have preclinical value in human prostate cancer chemotherapy.

A recent study mentioned a breakthrough in the discovery of a new HDAC inhibitor agent, the histone deacetylase inhibitor (S)-2 that induces apoptosis/differentiation in human prostate cancer cell lines (LNCaP and PC3). In the LNCaP cell line, (S)-2 was capable of triggering H3/H4 histone acetylation and H2AX phosphorylation, which are both DNA damage markers. Identically, (S)-2 was capable of stopping G0/G1 phase in cellular cycle. Again, (S)-2 leads to enhanced expression of both the protein and mRNA p21 levels in prostate cancer cells [84] . In the same way, HDAC molecules interact with the PRC2 complex, so that when cancer cells are treated with HDAC inhibitors such as SAHA, the PRC2 action would be counteracted, resulting in an increased acetylation at the precise location of loci, favoring gene expression. Similarly, a new HDAC inhibitor, the romidepsin was recently identified that inhibits HDAC1, reducing the binding of DNMTA and histone methyltransferase to the promoter region of tumor suppressor gene. Possibly, romidepsin would reduce H3K27 methylation level by affecting EZH2 and preventing the DNMT1 recruitment from the GSTP1 promoter. Other hypothesis is that romidepsin would eventually induce JMJD3 and UTX expressions, which are the specific histone H3K27 demethylases, causing then a fall in H3K27me3 levels in prostate cancer cells [85] . HMT inhibitors

The development of histone demethylating agents is of great interest, although actually very few molecules have been tested in clinical trials. HMT inhibitors are

Executive summary Prostate cancer • Prostate cancer is the most common cancer in men wherein occurs genetic mutations and epigenetic alterations.

DNA methylation inhibitors • The process of DNA methylation is carried out by DNA methyltransferase which adds a methyl group from a donor S-adenosyl-methionine to the 5 position of the cytosine ring mainly in the CpG dinucleotides. This reaction can be blocked by the drug: the nucleoside analog 5-aza-cytidine and 5-aza-2′-deoxycytidine which trap DNMTs at the DNA and inhibit propagation of DNA methylation during S-phase.

Histone modifications • N-terminal tails of histones are subject to a large number of post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deimination and proline isomerization. Among these modifications, histone acetylation and methylation play crucial roles in regulating gene expression and are associated with prostate cancer carcinogenesis.

Histone methyltransferase inhibitors • Promising preclinical assay in prostate cancer has been obtained with 3-deazaneplanocin A (DZNep) which was reported to selectively inhibit H3K27me3. DZNep is structurally analogous to sinefungin and adenosine dialdehyde.

EZH2 • Enhancer of zeste homolog 2 is the catalytic member of the PRC2 complex which catalyzes trimethylation of H3K27me3. EZH2 induces epigenetic silencing which subsequently results in tumorigenesis and metastasis. Overexpression of EZH2 is associated with tumor progression and poor prognosis in prostate cancer.

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a novel class of epigenetic drugs that have been recently described. Adenosine dialdehyde demonstrated diverse effects, including a decrease of H3K9 discovered and intended to trigger the demethylation of silenced genes in prostate cancer. Actually, a small molecule inhibitor of PRC2 called 3-deazaneplanocin A (DZNep) demonstrated great potential anti-tumor activity both in vitro and in vivo. DZNep is an inhibitor of S-adenosylhomocysteine hydrolase that catalyzes the conversion of adenosylhomocysteine to adenosine and homocysteine. Adenosylhomocysteine is produced by methyltransferase from the methyl donor S-adenosylhomocysteine, which inhibits methyltransferase leading to their degradation [86] . DZNep is strong enough to inhibit H3K27me3 and EZH2 expression in several cancer cell lines [87] . It is also well documented that DZNep could reactivate a set of PRC2 target genes in cancer cells independent of promoter DNA methylation [88] . Interestingly, DZNep was described to inhibit cancer cell invasion and tumor angiogenesis in prostate cancer. Additionally, two promising new HMT inhibitors, sinefungin and adenosine dialdehyde have been methylation, a suppression of AR expression and an inhibition of prostate cancer cell growth [89] . As a result, histone methyltransferase inhibitors might be an interesting strategy for the treatment of prostate cancer and could be chosen to provide clinical trials in combination with chemotherapy treatment. Yet, more investigations are needed in order to further study the development of new HMT inhibitor actions.

Conclusion & future perspective In this review, we focused on the different pathways in which epigenetic changes have been involved in molecular processes that control critical cellular mechanisms. Since the combination of DNMT, HMT, HAT and HDAC enzymes is crucial, their deregulation often leads to disease and malignancy. However, these enzymes constitute valuable therapeutic targets. Besides, next-generation sequencing technologies allowed providing information on epigenetic alterations at a large scale, helping identify several new epigenetic targets that enable the development of epigenetic therapies in prostate cancer. Furthermore, the chance to develop new techniques for biomarkers identification is greater and it would constitute powerful tools for prostate cancer detection, screening, diagnosis and treatment. Financial & competing interests disclosure The present paper was supported by grants from La Ligue contre le Cancer - Comites de la region Auvergne. S KarsliCeppioglu was supported by The Scientific and Technology Research Council of Turkey (TUBITAK-2219) project grants and A Dagdemir was supported by Protema Saglik Hizm. A.S. The authors declare that they have no competing interests. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. on gene transcription in prostate cancer. Am. J. Pathol. 183(6), 1960–1970 (2013).

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