Histone Modifications, Stem Cells and Prostate ...

Send Orders for Reprints to [email protected] Current Pharmaceutical Design, 2014, 20, 000-000

1

Histone Modifications, Stem Cells and Prostate Cancer Francesco Crea1,*, Pier-Luc Clermont1, Antonello Mai2 and Cheryl D. Helgason1,* 1 2

Experimental Therapeutics, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, Canada, V5Z 1L3; Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro, 5, 00185, Rome, Italy Abstract: Prostate cancer (PCa) is a very common neoplasm, which is generally treated by chemo-, radio-, and/or hormonal-therapy. After a variable time, PCa becomes resistant to conventional treatment, leading to patient death. Prostate tumor-initiating cells (TICs) and cancer repopulating cells (CRCs) are stem-like populations, driving respectively cancer initiation and progression. Histone modifiers (HMs) control gene expression in normal and cancer cells, thereby orchestrating key physiological and pathological processes. In particular, Polycomb group genes (PcGs) are a set of HMs crucial for lineage-specific gene silencing and stem cell self renewal. PcG products are organized into two main Polycomb Repressive Complexes (PRCs). At specific loci, PRC2 catalyzes histone H3 Lys27 trimethylation, which triggers gene silencing by recruiting PRC1, histone deacetylases and DNA methyl transferases. PRC1 catalyzes addition of the repressive mark histone H2A ubiquitination. Recently, the catalytic component of PRC1 (BMI1) was shown to play critical roles in prostate CRC self-renewal and resistance to chemotherapy, resulting in poorer prognosis. Similarly, pharmacological disruption of PRC2 by a small molecule inhibitor reduced the tumorigenicity and metastatic potential of prostate CRCs. Along with PcGs, some histone lysine demethylases (KDMs) are emerging as critical regulators of TIC/CRC biology. KDMs may be inhibited by specific small molecules, some of which display antitumor activity in PCa cells at micromolar concentrations. Since epigenetic gene regulation is crucial for stem cell biology, exploring the role of HMs in prostate cancer is a promising path that may lead to novel treatments.

Keywords: Epigenetics, polycomb, prostate, DZNeP, androgen receptor, LSD1. INTRODUCTION: TWO EMERGING PARADIGMS When Weinberg and coworkers demonstrated that specific DNA fragments are able to transform mouse fibroblasts into cancer cells [1], the idea that cancer was essentially a genetic disease opened a breach into the scientific community. This paradigm has become dominant for the following decades, leading to the identification of several oncogenes and tumor suppressor genes, whose mutations were thought to drive each step of cancer progression [2]. This model describes neoplastic progression as a microevolutionary process, with random genetic mutations occurring in a population of transforming cells and leading to the emergence of “dominant” clones with specific adaptive features [mobility, invasiveness, drug resistance) [3]. Following this assumption, cancer scientists discovered key pathways related to cancer progression [4], and developed some “biological” drugs targeting one or more pathways [5, 6]. Apart from some notable exceptions [7], those “magical bullets” targeting the Achille’s heal of cancer often failed in clinical trials [8, 9]. Even one of the most widely employed drugs, cetuximab, did not prove to prolong overall survival in genetically selected cancer patients [10], and some editorials questioned the possibility to significantly improve cancer patients’ survival in the next years [11]. In the last decade, two novel perspectives emerged to integrate the dominant model of cancer progression. First, it is now evident that, in addition to genetic changes, neoplasms are driven by epigenetic alterations [12]. Epigenetics refers to all heritable gene expression patterns, which are not due to changes of DNA primary structure [13], and in particular to DNA methylation (i.e. gene silencing) and histone post-translational modifications (HPTMs which may activate or repress gene expression). For example, *Address correspondence to these authors at the Experimental Therapeutics, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, Canada, V5Z 1L3; Tel: 604-675-800-7010. Fax: 604-675-8019; E-mail: [email protected] Tel: 604-675-8011; Fax: 604-675-8019; Email: [email protected] 1381-6128/14 $58.00+.00

epigenetic gene silencing of key oncosuppressor genes has been identified as an early event of oncogene addiction to cancer pathways, which often precedes somatic mutations [14]. DNA methylation patterns are widely altered in human cancers, showing global demethylation of non-coding regions [15], coupled with hypermethylation at specific loci [16]. More recently, HPTMs emerged as crucial regulators of cancer cell behavior [17]. Interestingly, epigenetic alterations are reversible, and can be targeted by specific drugs. Thus, identifying key epigenetic pathways in cancer cells might pave the way to innovative therapeutic approaches [18]. The other emerging paradigm is the hierarchic model of cancer initiation and progression. Pre-clinical evidence originally from a leukemia model, and subsequently expanded to many solid tumors, indicates that cancer is not a “democratic” microevolutionary process [19]. Indeed, only a small percentage of the total tumor mass is able to initiate the tumor and sustain cancer progression. Since prostate tumor initiation and progression seem to be triggered by two different populations with stem-like phenotype, we will not use the term “cancer stem cells”, which might be confounding. As previously proposed [20], we will distinguish tumor initiating cells (TICs) from cancer repopulating cells (CRCs). The first population identifies the cell that, acquiring a set of genetic and epigenetic alterations, gives rise to PCa. The latter is the only cancer cell fraction able to reconstitute the entire tumor of origin, when injected into immunocompromised mice [21]. CRCs are also responsible for drug resistance and metastatic spreading, and thus are very interesting for translational research [19]. For this reason, we will particularly focus on this sub-population. Interestingly, epigenetic gene regulation seems to be crucial for CRC biology [22, 23], thereby providing a link between those two innovative theories. In the present manuscript, we will summarize current knowledge on epigenetic regulation of CRCs, with particular regard to HPTMs. We will focus on prostate cancer (PCa), which is the most common male malignant neoplasm in developed countries [24], and is one of the most investigated cancer types in both epigenetic [25] © 2014 Bentham Science Publishers

2 Current Pharmaceutical Design, 2014, Vol. 20, No. 00

and TIC/CRC [25] research fields. We will then illustrate some molecules which might target epigenetic pathways in prostate CRCs, with particular emphasis on their possible clinical application. STEM CELL MODEL OF PROSTATE CANCER INITIATION AND PROGRESSION Prostate Cancer Initiation PCa is a leading cause of morbidity and mortality worldwide, and in the U.S. alone it affects more than 240,000 men each year [24]. This widespread neoplasm usually begins as an indolent and localized disease, which can be treated by radical prostatectomy and/or radiotherapy [26]. A significant fraction of those patients (15-33%) progress to metastatic disease [27]. Metastatic PCa is generally treated with drugs that disrupt androgen receptor (AR) signaling, which is a driving pathway of prostate cells’ proliferation. Despite this, almost all metastatic patients will develop a castration-resistant prostate cancer (CRPCa), which leads to death within a few months [28]. CRPCa cells adapt to the low-androgen environment by AR over-expression, ligand-independent activation or aberrant up-regulation of co-regulators [29]. Despite the introduction of novel therapeutic agents, which delay metastatic PCa progression [27, 30], evolution to a metastatic and castrationindependent status is still unrestrainable. Thus, the identification of novel targets, and development of effective therapeutics to target them, is still needed in this field. For this reason, we think that exploring the biological roots of PCa may indicate new strategies of treatment. Normal prostate epithelium is composed of three main layers (Fig. 1) [20, 31]: 1) basal cells, which are located close to the basal membrane, are AR-negative and express cytokeratins 5 and 14 (K5, K14); 2) luminal secretory cells, expressing K8, K18, AR and secreting prostate specific antigen (PSA) in response to androgen signaling; 3) neuroendocrine cells, a rare sub-population of paracrine cells, which express specific markers, such as chromogranin-A (CHGA), synaptophisin (SYP), and neuron specific enolase (NES) [32, 33] . Prostate SCs are defined as cells capable of long-term self renewal and differentiation into all mature prostate cell types. Seminal data indicate that a rare population of SCs is able to fully reconstitute the adult organ in both murine [34] and human [35] prostate. The SC compartment accounts for a very small fraction (less than 1%) of total prostate cells [36], and is traceable through both surface and intracellular markers, including CD133, CD49f, Trop2 and p63 [35, 37, 38]. The specific cellular role of those markers in SC biology is emerging through functional studies. For example, p63 seems to be required for early steps of SC commitment, since it is essential for prostate tissue specification in the urogenital sinus [38]. As reviewed by Taylor and co-workers [20], it is still controversial whether prostate SCs originate from the basal or luminal layer. It is likely that both compartments hold a small fraction of SCs: basal SCs are AR-, while luminal SCs are AR+, but both types can survive after androgen ablation (castration resistance). In each case, normal prostate demonstrates a hierarchical organization, with a small population of slow-cycling, self-renewing and pluripotent SCs which is able of reconstituting all mature cell types (Fig. 1). Prostate Cancer Repopulating Cells PCa might originate from aberration of physiological developmental mechanisms [39]. Following this assumption, it is not surprising to find that PCa is often composed of a variable mixture of cells with luminal, neuroendocrine and stem-like phenotypes. A common finding of PCa specimens is the loss of basal markers [40]. According to this evidence, seminal works indicated that luminal

Crea et al.

SCs are able to transform into PCa cells, upon activation of key signaling pathways (AR, Akt, Myc) [41, 42]. Recent works also identified a potential reservoir of neoplastic transformation in the basal layer. In particular, transduction of basal prostate cells with activated Akt, Erg (an oncogenic transcription factor) and AR resulted in basal cell transformation and generation of PCa in vivo [43]. Although luminal TICs generally show SC features, a recent report indicates that basal CD133- cells are more tumorigenic than CD133+ cells, possibly due to their higher proliferative activity [36]. Thus, PCa might originate from basal and luminal SCs, as well as from transient amplifying cells, acquiring a set of molecular alterations. This model is similar to the multi-cellular carcinogenesis paradigm of breast cancer initiation [44]. Interestingly, molecular pathways of PCa initiation seem to be at least in part shared by luminal and basal TICs. For example, knockdown of the Pten phosphatase, which in turn activates Akt, is able to trigger neoplastic transformation in both luminal [41] and p63+ basal cells [45]. Notably, Pten gene inactivation by both genetic and epigenetic hits is a common finding in human PCa cells [46, 47]. It is worth noting that a variable percentage of PCa cells also express neuroendocrine markers, and that neuroendocrine differentiation is a poor prognostic factor in PCa [48]. Thus, it is conceivable that neuroendocrine cells might contribute to PCa initiation, in addition to basal and luminal cells. In summary, PCa TICs may reside in all normal prostate cell types, and PCa is generated by SCs (or transient amplifying cells) acquiring genetic and epigenetic alterations (Fig. 1). Independently of the cell of origin, PCa itself shows a hierarchical organization, which mirrors normal tissue architecture. Collins and co-workers first identified a sub-population of CD44+/alpha2beta1Integrinhi/CD133+ PCa cells with SC features [49]. It has been shown that this fraction expresses basal markers and is AR-.. They represent 0.1% of the total tumor mass, but are the only cells able to sustain in vitro self-renewal, and to generate differentiated cancer cells (DCCs), expressing AR and other luminal markers. DCCs, representing the vast majority of cancer cells, are not capable of limitless self-renewal. However, CRCs are defined by the ability to regenerate the original tumor through several in vivo passages [19]. Thus Collin’s seminal results just suggested the existence of a CRC fraction in PCa. A few years later, Patrawala et al. found a population of PCa cells with complete CRC features (self-renewal, in vivo tumorigenicity, pluripotence), just separating CD44+ PCa cells from human xenografts [50]. Subsequently, Hurt and co-workers [51] identified a similarly rare CRC subpopulation in commonly used PCa cell lines. This CRC fraction was identified through the CD44+/CD24-phenotype. Both Patrwala and Hurt demonstrated that their CRC fraction is able to reconstitute a phenocopy of the original tumor in vivo, and that the “depleted” fraction has no tumorigenic activity. Like neural SCs, prostate CRCs are able to form spheres when cultured in serumreplacement medium [52]. A particular component of human serum (vitronectin) is able to trigger “prostatosphere” (PS) differentiation [53]. It is worth noting that there is no evidence showing a direct relationship between prostate CRCs and the cell(s) of origin of PCa. Due to their ability to reconstitute in vivo the whole tumor mass, CRCs are thought to be a stem-like population, which is able to sustain cancer growth [20, 21]. Some commentaries have argued that CRCs might simply reflect a higher adaptive and tumorigenic potential in murine environment [54]. Nonetheless, pre-clinical and clinical data indicate that CRCs are a very interesting subpopulation in PCa, and a possible therapy target [55]. Indeed, emerging evidence indicates that CRCs might be the seeds of metastatic spreading and drug resistance. CRCs selected for the CD44+/CD24- phenotype are significantly more invasive than DCCs [50, 56]. Concurrent activation of Akt- and Ras-dependent pathways may explain at least in part the higher metastatic potential of CRCs [57]. In addition, some soluble factors might orchestrate

Histone Modifications and Prostate Cancer

Current Pharmaceutical Design, 2014, Vol. 20, No. 00

3

Fig. (1). Prostate cancer tumorigenesis and progression (the stem cell model). Normal prostate is composed of basal (BC), luminal (LC) and neuroendocrine cells (NE). All three cell types originate from normal stem cells (SC). Prostate cancer might originate from SCs located in basal or luminal layers. An alternative model suggests that CD133- BCs are highly tumorigenic. CRCs (Cancer repopulating cells) are a small fraction of PCa cells. CRCs retain some SC features, are the only long-term proliferating cancer cells, which are able to self-renew, or produce differentiated cancer cells (DCC), i.e. the bulk tumor mass. CRCs are considered responsible for drug resistance and metastatic spreading.

CRC metastatic potential. For example, it has been shown that bone morphogenetic protein-7 (BMP7) induces CRC senescence, displaying anti-metastatic activity [58]. In a mouse model of PCa, BMP7 withdrawal results in CRC expansion and macroscopic metastatic disease. Keeping with this pre-clinical model, lower expression of BMP receptor-2 in PCa specimens correlates with longer relapse-free survival after prostatectomy [58]. It is still controversial whether all CRCs trigger metastasis, or if only a subset of those rare cells is able to diffuse through the bloodstream and grow in distant organs [59]. A further interesting peculiarity of prostate CRCs is to survive (almost) all current treatments, thereby providing a reservoir for cancer recurrence. PSA-/low cells isolated from PCa display selfrenewal and tumorigenic properties of CRCs, and express further CRC markers (CD44, alpha2beta1Integrin) [60]. In mouse models, androgen deprivation selectively spares the PSA-/low fraction, which in turn generates a castration-resistant tumor. The PSA-/low fraction is also more resistant to taxanes than the PSA+ one, partly because it is mostly composed of cells in the G0 phase. Interestingly, the SC factor NANOG, which is expressed by prostate CRCs, is able to induce castration-resistance in AR+ PCa cells [61]. Prostate CRCs display selective up-regulation of growth pathways, including Aktand Wnt- dependent signaling, which can mediate chemo-resistance [22]. It has been shown that CRCs are more resistant than DCCs to docetaxel, and that a combined therapy with Akt inhibitor and chemotherapy may eradicate both PCa subpopulations [62]. Finally, PCa cells’ irradiation seems to induce long-term recovery of prostate CRCs, thereby providing a plausible mechanism for delayed tumor recurrence after radiotherapy [63]. Whole genome gene expression profiling of human cancers revealed that activation of SC pathways (including NANOG, SOX2 and BMI1) predicts poor prognosis and therapy resistance in some epithelial neoplasms, including PCa [64, 65]. Finally, CRCs have recently displayed an unsuspected level of pluripotency in some brain tumors. De Maria and coworkers [66]

demonstrated that glioblastoma CRCs are also able to differentiate into endothelial cells, thereby driving neoangiogenesis Although this phenomenon has not been investigated in PCa, it is intriguing to speculate a similar mechanism for this tumor, where neovascularization plays a peculiar role in patients’ prognsosis [67]. In conclusion, the relationship between prostate SCs, TICs and CRCs remains obscure. Despite this, we know that both normal prostate and PCa share a hierarchical organization, and that targeting CRCs might prevent metastasis and drug resistance, thereby eradicating PCa. HISTONE POST-TRANSLATIONAL MODIFICATIONS AND PROSTATE CANCER REPOPULATING CELLS The Histone Code Epigenetic misregulation represents a key driver in the initiation and progression of PCa [68-70]. In particular, histone posttranslational modifications (HPTMs) play a major role in establishing and maintaining PCa CRCs (summarized in Fig. 2) [71]. In nonmitotic cells, histones and DNA are packaged into nucleosomes. In each nucleosome, about 150 bp of DNA wrap around a core of histone proteins made up of an H3-H4 tetramer and two H2A-H2B dimers [72]. Histone N-terminal tails protrude from the core structure and can be chemically modified by a wide variety of enzymes. The synergistic effect of these chemical modifications determines whether the chromatin will adopt a transcriptionally active (euchromatin) or inactive (heterochromatin) state [73]. Three general types of enzymes mediate epigenetic regulation of transcription through HPTMs: the ‘‘writers’’, ‘‘the erasers’’, and the ‘‘readers’’ [74]. ‘‘Writers’’ covalently add chemical groups to histones in a highly regulated fashion. Three of the best-characterized HPTMs include acetylation, methylation, and ubiquitination, which are catalyzed respectively by histone acetyltransferases (HATs), histone lysine methyltransferases (HKMTs), and E3 ubiquitin ligases [75, 76]. Other reported HPTMs include sumoylation, phosphorylation, ADP-ribosylation, citrullination, and biotinylation [77]. This


diverse set of ‘‘writers’’ allow the simultaneous existence of an astronomically high number of HPTM combinations. Interestingly, all of these HPTMs are reversible due to the existence of ‘‘erasers’’, which mediate the removal of the aforementioned chemical modifications [74]. To date, histone deacetylases (HDACs) constitute the most widely studied ‘‘erasers’’, but recent studies have revealed the importance of lysine demethylases (KDMs) in SC and cancer biology [78]. The third type of enzymes, the ‘‘readers’’, do not modify histones directly but recognize specific HPTMs and act as docks for other complexes that remodel the three-dimensional chromatin structure in an ATP-dependent fashion (Fig. 2) [79]. Histone Acetylation In 1964, Allfrey et al. demonstrated that acetyl groups could be enzymatically added to histone tails post-translationally and that acetylation correlates with an increase in transcriptional competency [80]. Histone acetyltransferases (HATs) catalyze the acetylation of lysine residues on histone tails, thereby neutralizing the positive charge on the lysine and reducing the electrostatic affinity between histones and the negatively-charged DNA. Genes located near acetylated histones consequently tend to be highly expressed as they are more readily accessible to the transcription machinery [81]. Acetyl groups can be removed from histones by ‘‘eraser’’ proteins named histone deacetylases (HDACs) which antagonize the effect of HATs. Complexes with deacetylase activity repress the expression of their transcriptional targets [82]. Recent studies show that aberrant regulation of histone acetylation plays a role in the malignant progression of PCa. For example, the histone acetyltransferases p300 and cAMP-responsive element-binding protein (CREB) are overexpressed in a number of prostate tumors and their preponderance correlates positively with tumor volume, prolifera-

Crea et al.

tion markers, and Gleason score [83]. Recent data suggest that the oncogenic effect of p300 results from cooperation with AR. P300 increases the acetylation levels of histones H3 and H4 at androgenresponsive elements (AREs) near the transcriptional start site of AR target genes. Ianculescu et al. demonstrated that p300 is required for the expression of a large number of AR-regulated genes in C42B, an advanced but AR+ PCa cell line [83]. Since the PCa CRCs are not dependent on AR activation [60], the activity of p300 most likely plays a very limited role in CRCs. This implies that the cooperative effect of p300 and AR likely occurs in more differentiated PCa cells [20]. As for HATs, the role of HDACs in PCa remains poorly characterized. Previous studies have reported increased deacetylase activity in PCa [84]. HDACs have also been associated with the silencing of DAB2IP in PCa cell lines [85]. However, attempts to further characterize the role of HDAC in PCa and PCa CRCs did not yield any consistent results. Furthermore, HDAC inhibitors had little to no effect in phase 1 and phase 2 clinical trials [86-88], and resulted in high levels of toxicity. In summary, histone acetylation does not seem to represent a central element of epigenetic regulation in PCa CRCs. Histone Methylation In the early 2000s, Rea et al. identified a functional interdependence of site-specific H3 tail methylation marks from which arises a dynamic mechanism that regulates higher-order chromatin structure [85]. Multiple studies have followed and have shed light on the mechanism and biochemical significance of histone methylation. Many histone lysine methyltransferases (HKMTs) have been identified, allowing the catalysis of mono-, di-, or tri-methylation of lysines on histone tails [71]. These modifications can trigger the formation of either transcriptionally active euchromatin or tran-

Fig. (2). Epigenetic gene regulation and CRC biology. The nucleosome is composed of DNA (thin black line) wrapped around core histones (represented by green circles). Epigenetic alterations include histone modifications and DNA methylation (blue rectangles and triangle, respectively) and are mediated by specific enzymes or complexes (orange circles). Some of those epigenetic regulators contribute to CRC features (metastasis, chemoresistance, self-renewal). Acronyms are described in the text. Modified from Crea et al. Mol Cancer 11:52 (2012).


scriptionally inactive heterochromatin depending on the specific methylated residue, the number of methyl groups added and the availability of factors that remodel chromatin. For example, trimethylation of lysine 4 (H3K4me3) is catalyzed by members of the trithorax family and results in transcriptional activation of nearby genes [89]. HKMTs such as SUV39H1, Clr4p, DIM-5, Su(var)3-9 or SUVH2 methylate H3K9 and consequently trigger heterochromatin assembly and transcriptional repression [90]. The polycomb group (PcG) of proteins represent another family of histone modifiers that mediate repressive epigenetic events. PcG products are organized into two main Polycomb Repressive Complexes (PRCs). PRC2 trimethylates H3 Lysine 27 (H3K27me3), an epigenetic event associated with transcriptional repression [91]. PRC2 is comprised of at least EZH2, Eed, and Suz12. EZH2 represents the catalytic subunit of PRC2 and its methylase activity occurs through its SET domain. H3K27me3 recruits the PRC1 complex to specific loci via an interaction mediated by members of the CBX family of proteins [92]. BMI1 and the E3 ubiquitin ligase Ring1b add ubiquitin moieties to lysine 119 on H2 thereby accounting for the catalytic activity of PRC1. H2K119Ub represents a strong repressive mark which cooperates with H3K27me3 to silence transcription. Although there is strong evidence for the synergistic effect of PRC1 and PRC2, it has been demonstrated that the two complexes can also silence genes independently of each other [93]. Moreover, PRC2 also recruits DNA methyltransferases (DNMTs) and HDACs to accentuate the repressive chromatin state of its targets [94, 95]. These findings suggest that PcG proteins control the establishment of a complex epigenetic network that regulates gene transcription. Ever since their discovery as transcriptional regulators of Homeobox genes, PcG have been linked to molecular pathways responsible for SC differentiation and self-renewal [96]. An increasing number of studies demonstrate that PRC1 and PRC2 silence lineage-specification genes in self-renewing embryonic SCs (ESCs), but are also essential to the repression of pluripotency genes during ESC differentiation [97]. Moreover, knock out of either EZH2 or Ring1b results in early embryonic lethality in mice [98, 99]. PcG also play an important role in prostate development, as well as in the maintenance of the adult prostate SC pool. In vitro, BMI1 controls prostate SC self-renewal activity and is required for normal prostate tubule regeneration in vivo [100]. Interestingly, overexpression of both EZH2 and BMI1 was reported in prostate tumors and correlated with an aggressive phenotype [101]. Yu et al. developed a ‘‘Polycomb repression signature’’ composed of 14 direct targets of PcG in metastatic tumors that predicted poor clinical outcome in multiple microarray data sets, including PCa [102]. Interestingly, the genes repressed in the ‘‘Polycomb repression signature’’ in metastatic PCa are also silenced in ESCs. Those results support the idea that the aggressiveness of a prostate tumor depends on a subpopulation of tumor cells which have acquired stem-like properties as a result of epigenetic alterations. We chose to refer to these cells as PCa CRCs due to their ability to reconstitute the entire tumor of origin when injected into immunocompromised mice and to form PS when cultured in serum-replacement medium [20]. As mentioned above, CRCs have been isolated based on the expression of specific markers, and are considered the seeds of clinically relevant events (metastasis, recurrence after therapy) [55]. The aggressive behavior of PCa CRCs can be attributed to key epigenetic changes that initiate a transcriptional program promoting invasion which is highly reminiscent of the epithelial-mesenchymal transition (EMT) [57]. Many reports indicate that EZH2 represents a key driver in the progression of PCa. 3-Dezaneplanocin-A (DZNeP) is an Sadenosyl-L homocysteine hydrolase inhibitor which impairs the catalytic activity of EZH2 [103]. DZNeP is effective both in vitro and in vivo against PCa cells. Pharmacologic disruption of PRC2 with DZNeP inhibits PS formation and reduces the CD44+/CD24fraction in PCa cell lines, thereby hindering in vivo tumorigenicity


5

[104]. EZH2 also acts by regulating multiple pathways associated with CRCs. First, EZH2 cooperates with HDAC and SNAIL to repress CDH1 expression, a critical step for EMT [105]. Moreover, PRC2 silences the CDKN2A and CDKN2B loci encoding the tumor suppressors p14 (ARF), p15 (INK4B) and p16 (INK4A) [106], thereby providing a mechanism for long-term CRC self-renewal. The anti-metastatic gene DAB2IP represents another target of EZH2 in PCa. DAB2IP is a potent cell growth inhibitor and modulator of Ras-signalling, a pathway closely linked to metastasis [85]. Overexpressed EZH2 recruits HDACs at the DAB2IP promoter, which triggers metastasis. Metastatic PCa also shows a global increase in H3K27 methylation with respect to localized disease, which signifies that the PRC2 complex gets activated in the progression to metastasis [107]. EZH2 also silences two members of the tissue inhibitors of metalloproteinase, TIMP2 and TIMP3, an event that promotes angiogenesis and metastasis in PCa [108]. In endothelial cells of invasive ovarian cancer, VEGF activates the expression of EZH2, which subsequently represses VASH1, a known inhibitor of neovascularization [109]. Another key component of EZH2’s oncogenic potential lies in its ability to recruit DNMTs, an event possibly associated with drug resistance. ChIP analyses report binding of DNMTs to several PRC2-repressed genes which depends on the presence of EZH2 [110]. Furthermore, bisulphite genomic sequencing experiments demonstrate that EZH2 is required for DNA methylation of EZH2-target promoters. In PCa cell lines and in human tissues, treatment with the anti-androgen drug bicalutamide increases DNMT expression and activity which results in a castration resistant phenotype [111]. Since EZH2 regulates the recruitment of DNMTs to chromatin, induction of DNA methylation represents one mechanism by which PRC2 can mediate drug resistance. Gal-Yam et al. proposed a model to explain the interplay between PcG and DNMTs in PCa cell lines. Their model is characterized by a frequent switching of Polycomb repressive marks and DNA hypermethylation [112]. All those data suggests that PRC2 controls many important molecular pathways that maintain the invasiveness of CD44+/CD24- cells. An important question arises: What regulates the expression and activity of EZH2? While this question remains largely unanswered, recent studies provide insights into how EZH2 regulation occurs. ETS family transcription factors can either activate or repress the expression of EZH2 and about 80% of prostate tumors have one or more aberrantly expressed ETS gene [113]. For example, Erg activation increases the levels of EZH2 and H3K27me3. Notably, many prostate tumors contain a fusion protein resulting from a chromosomal rearrangement between TMPRSS and ERG [114]. Phosphorylation of EZH2 by CDK1 and CDK2 increase EZH2 recruitment to chromatin in a cell cycle-regulated manner [115]. Moreover, Akt kinase can phosphorylate serine 21of EZH2 which results in PRC2 inhibition [116]. An increasing number of studies report a role for tumor suppressing miRNAs in the regulation of PRC2 activity. Genomic loss of miR101 and miR-26a leads to EZH2 overexpression [117, 118]. Loss of let-7 also upregulates EZH2 and is associated with the acquisition of SC signatures [119]. Despite these insights, the regulation of PRC2 activity remains unclear and warrants further investigation. PRC1 represents the other PcG complex known to regulate PCa CRCs. PRC1 can be recruited to chromatin by recognizing H3K27me3 marks on histones and subsequently catalyzing the monoubiquitination of lysine 119 on H2A [71]. As a result, PRC2 and PRC1 synergistically cooperate to repress specific loci. PRC1 also functions independently of PRC2 [120]. For instance, loss of PRC2 in ESCs is associated with a global decrease in H3K27me3 but not in H2AK119Ub [121]. Proper PRC1-mediated repression requires the presence of a functional BMI1 subunit [122]. The activity of BMI1 is essential for the self-renewal capability of hematopoietic SCs [123]. Many reports indicate that BMI1 also drives the formation and maintenance of CRCs in many types of malignancies. For example, the proliferative potential of leukaemic CRCs lacking BMI1 is compromised in vitro and in vivo [124]. In laryn-


geal squamous cell carcinoma, BMI1 was crucial for tumor initiation in xenografts and its knockdown specifically repressed proliferation and promoted apoptosis of CD133+ cells [125]. MiR-200c suppresses clonogenicity of breast cancer CRCs through the downregulation of BMI1 [126]. Recent evidence suggests that BMI1 also critically regulates the self-renewal ofnormal and cancer stem cells in the prostateExpression of BMI1 is required for the formation of PS, which are enriched of CRCs [127]. A study by Lukacs et al. demonstrated that BMI1 crucially controls prostate SC self-renewal and malignant transformation [100]. Moreover, high BMI1 expression induces docetaxel chemoresistance in PCa cells [128]. Since BMI1 overexpression represents a hallmark of aggressive PCa, a mechanism by which BMI1 promotes the formation of PCa CRCs could explain why prostate tumors with high levels of BMI1 tend to have poorer prognosis. The majority of the genes repressed by BMI1 play a role in proliferation, self-renewal, and differentiation. BMI1 promotes PCa initiation by suppressing p16INK4A and p14ARF expression [129]. In addition, BMI1 activates Akt and hTERT (telomerase reverse transcriptase) via PTEN phosphatase repression [130]. Interestingly, Akt and hTERT are required for PCa CRC self-renewal [62, 131]. The pathways regulated by BMI1 suggest that the maintenance of PCa CRC critically depends on BMI1 integrity. Inhibiting BMI1 therefore represents an interesting therapeutic strategy for PCa treatment. While previous studies in epigenetics of PCa mainly focused on PcG, there is now much effort to characterize the role of histone lysine demethylases (KDMs). KDMs are ‘‘eraser’’ proteins which remove methyl groups from lysine residues on histones [78]. Depending on the specific residue, KDM activity can result in either transcriptional activation or repression. Increasing evidence demonstrates that KDMs play an important role in PCa progression [132]. In particular, LSD1 represents a new and promising drug target. High levels of LSD1 are found in aggressive PCa and correlate significantly with disease relapse [133]. LSD1’s main catalytic function involves the conversion of dimethylated H3K4 to monoand unmethylated H3K4 [134]. Since dimethylated H3K4 is associated with transcriptional activation, demethylation of H3K4me2 promoted by LSD1 leads to transcriptional repression at target loci [135]. LSD1 activity is highly dependent on cellular context and is modulated by interactions with multiple co-factors [136]. LSD1 interacts with HDACs to repress transcription of key sets of genes responsible for tumor suppression which leads to an increase in self-renewal [137]. In PCa, LSD1 demethylates H3K9me3 and enhances androgen-receptor-dependent transcription [138]. New studies suggest a role for LSD1 in CRCs. LSD1 inhibitors abolish the proliferation of stem-like cancer cells which express Oct4 and Sox2 in many malignancies [139]. Notably, these inhibitors did not affect the growth of differentiated cancer cells or normal somatic cells. Since LSD1 orchestrates many processes involved in PCa progression and has been associated with SC self-renewal, it is possible that LSD1 also critically promotes the CRC phenotype in PCa. It would be interesting to determine whether LSD1 cooperates with PcG to induce PCa CRCs, or if they function separately. In summary, HPTMs are emerging as crucial regulators of PCa CRCs, and might be targeted to prevent metastasis and drug resistance. While the role of PcGs is well established, the function of KDMs or other epigenetic modifiers should be clarified by future studies. Nonetheless, epigenetic regulation of PCa CRCs represents a critical aspect of PCa and requires further analysis. The discovery of factors that control HPTMs could lead to promising new treatments for aggressive PCa. DRUG TARGETING HISTONE MODIFICATIONS, POSSIBLE THERAPEUTIC IMPLICATIONS FOR PROSTATE CANCER Among therapeutic small molecules acting through an epigenetic mechanism (epi-drugs), DNMT and HDAC inhibitors are

Crea et al.

surely the most studied and validated in cancer. A number of clinical trials involving such compounds, alone or in combination with other chemotherapeutic agents, is actually ongoing to detect their capability to counteract hematological malignancies as well as solid tumors. With few notable exceptions, most pre-clinical studies investigate the effect of epi-drugs on total tumor cells, rather than on specific sub-populations (e.g. CRCs). Focusing on PCa, vorinostat (SAHA, approved in 2006 by the FDA for treatment of refractory cutaneous T-cell lymphoma) (Fig. 3) has been reported to be able to enhance radiation-induced cytotoxicity in DU145 cells [140], and showed synergy with zoledronic acid in inducing cell death in LNCaP and PC3 cells [141]. In combination with the AR antagonist bicalutamide, vorinostat inhibited cell proliferation in the androgenresponsive LNCaP cells but had no effect in the AR- PC3 PCa cells [142]. Trichostatin A (TSA) and sodium butyrate (Fig. 3) led to TRAIL-induced cell death in a panel of prostate tumor cell lines (ALVA-31, DU-145, and LNCaP) with varying TRAIL sensitivity [143], and were able to inhibit PC3 and LNCaP cell proliferation by down-regulating the telomerase activity via suppression of hTERT mRNA expression [144]. Sodium butyrate displayed growth inhibition and apoptosis in human PCa in vivo [145], and in LNCaP cells it induced the expression of AR in the nuclear compartment, significantly decreased the expression of the cell cycle regulatory proteins, and up-regulated p21Waf1/Cip1 and p27Kip1 after 48 h treatment [146]. Valproic acid (VPA, Fig. 3) is another short-chain fatty acid able to check cell proliferation, and to up-regulate AR levels and E-cadherin expression in human PCa cells. The activity of VPA is more pronounced in androgen independent PC3 cells than androgen dependent LNCaP cells, although growth inhibitory effects are detectable at quite elevated concentrations (0.45 mM) [147]. VPA pre-treatment is also able to enhance androgen responsiveness of C-81, C4-2 and MDA PCa2b-AI cells [148]. More importantly, VPA was shown to induce unfavorable neuroendocrine transdifferentiation (NET) in PC3 cells both in vitro and in vivo [149]. In the case of PCa progression, the cells showing NE phenotypes with NE markers are increased, and correlated with poor prognosis and androgen-independence. The NET process induced by VPA was related to Bcl-2 over-expression in non-NE PCa cells and to activation of PPAR in NE cells, and the use of specific PPAR antagonists was able to reduce significantly the expression of NE markers induced by VPA. Because of the dual effect of VPA as a HDAC inhibitor as well as inducer of NET, the NET pathway needs to be blocked while using VPA for therapy of PCa. Two hydroxamate HDAC inhibitors highly involved in clinical trials, belinostat and panobinostat (Fig. 3), have been recently studied also in PCa. Treatment of LAPC-4 and 22rv1 (androgen-dependent and expressing AR) and PC3 (androgen-independent not expressing AR) cells with belinostat showed that this HDAC inhibitor preferentially elicited antitumor activity in androgen-dependent tumor cells expressing AR, by induction of p21 and p27, acetylation of p53 and G2/M arrest associated with Bcl2 and Bcl-Xl downmodulation, significant reduction of survivin and phosphorylated Akt and increased caspase-8 and -9 expression/activity [150]. Notably, this study demonstrated that belinostat is active at micromolar concentration on AR+ but not on AR- PCa cells. Those data, along with poor activity and relative toxicity displayed by HDAC inhibitors in phase 1 and 2 clinical trials [86, 88], indicates that HDAC inhibitors might not be the optimal epi-drugs for targeting PCa CRCs. One possible explanation is that HDAC seems not to play a prominent role in CRC self-renewal. Keeping with data on PCa epigenetics, HDAC inhibitors seem to be more active on “differentiated” cancer cells, which express AR. Thus, the identification of novel targets, active in androgen-independent CRCs, may provide new tools for oncologists. As already outlined, PcGs are crucial for CRC self-renewal, and are involved in metastatic spreading, drug resistance and angiogenesis. The PcG member EZH2 is increasingly expressed during


Current Pharmaceutical Design, 2014, Vol. 20, No. 00 O

O

H N

N H

O

H 3C

TSA HDAC inhibitor

OH

N H

H 3C

ONa

VPA HDAC inhibitor

sodium butyrate HDAC inhibitor

O2 S

CONHOH

belinostat HDAC inhibitor NH 2

CONHOH H N

N

N

N CH3

N CH 3

N H

HO

pargyline LSD1 inhibitor

panobinostat HDAC inhibitor

OH OH DZNep EZH2 inhibitor

O O2 N

Cl O

NH 2

CF3

tranylcypromine LSD1 inhibitor

namoline LSD1 inhibitor

N H

O O

NH 2

O O

HN

NCL-1 LSD1 inhibitor

O NH 2

N

O O

N

HN

H N

O

NH2 N

OH

O

O H 3C

N H

CH3 CH3

N CH 3

vorinostat HDAC inhibitor

O

OH H 3C

7

OH

H 3CO

HO

NCL-2 LSD1 inhibitor

O

OCH3 curcumin HAT inhibitor

HO

OH

OH OH O

decitabine DNMT inhibitor

O O

H3 C

O NHOH

N H

H N

N

N H CH3

entinostat HDAC inhibitor

AR42 HDAC inhibitor

NH 2

O

O H 3C

N

N

CH 3

OH COOH

hydroxamate KDM4A/KDM4C inhibitor

Fig. (3). Chemical structures of epi-drugs described in the present manuscript. Under each compound, the target enzyme is specified. Acronyms are described in the text.

PCa progression, and data from PCa cells transfected with EZH2 small interfering RNA demonstrated that reduced EZH2 levels resulted in the dissociation of the EZH2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter [85]. Consistently, non toxic concentrations of the EZH2 inhibitor DZNeP (Fig. 3) completely eradicated LNCaP (AR+) and DU145 (AR-) PS formation, and significantly reduced the expression of CRC markers [104]. In the same conditions, non toxic concentrations of 5-aza-2'-deoxycytidine (decitabine) (Fig. 3) did not affect PS formation, and TSA eradicated PS in LNCaP, but not in DU145 cells. This evidence suggests that DZNeP might be more effective than HDAC and DNMT inhibitors in eradicating PCa CRCs, particularly on androgen independent cells. Due to the aforementioned role of EZH2 in EMT and tumor diffusion, DZNeP might be particularly useful to prevent metastatic spreading. Along

with its promising activity, DZNeP holds some drawbacks: it is for example a very hydrophilic molecule, which is readily cleared from bloodstream. Thus, its exploitation in the clinical setting has been questioned [151]. However, recent data indicate that a pegylated liposomal formulation of DZNeP displays a significantly better pharmacokinetic profile and longer half-life [152]. A further strategy to target CRCs could be affecting multiple epigenetic pathways involved in CRC biology (Fig. 2). Curcumin (Fig. 3) is an HAT p300 inhibitor known as a potent anticancer phytochemical. Treatment of LNCaP cells with curcumin led to demethylation of the first 14 CpG sites of the CpG island of the Neurog1 gene and restored the expression of this cancer-related CpG-methylation marker gene [153]. In these experiments, curcumin dramatically decreased MeCP2 (methyl CpG binding protein 2, a transcriptional repressor) binding to the promoter of Neurog1,


inhibited total HDAC activity with differences between the single HDAC isoforms, and reduced the levels of H3K27me3 (a marker of EZH2 activity) at the Neurog1 promoter region as well as at the global level. These data seem to suggest that curcumin may be a multi-target epigenetic modifier, able to reactivate epigenetically silenced genes in human PCa cells. The specific activity of such multi target epi-drugs on CRCs needs to be clarified. The H3K4 histone demethylase LSD1 seems to be a validated target to inhibit in PCa, and might be involved in CRC biology, as already outlined. After the first study claiming pargyline (Fig. 3), a well-known monoamine oxidase (MAO) inhibitor, as a LSD1 inhibitor active in PCa cells after AR induction [138], and then not confirmed by other authors [154, 155], a second MAO inhibitor, tranylcypromine (Fig. 3), proved to be more efficient in inhibiting H3K4 demethylation by LSD1, and two tranylcypromine-based compounds, NCL-1 and NCL-2 (Fig. 3), showed GI50 (50% growth inhibiting concentration) values ranging from 6 to 17 micromolar in androgen independent PCa cells [156]. Very recently, the –pyrone namoline (Fig. 3) has been described as a reversible LSD1 inhibitor able to inhibit LNCaP cell proliferation, expression of AR target genes FKBP5 and TMPRSS2, and tumor growth in a mouse PCa model [157]. H3K4 demethylation was also suppressed by treatment of LNCaP cells with HDAC inhibitors. In particular AR42, entinostat, and vorinostat (Fig. 3) led to up-regulation of H3K4 methylation by decreasing the expression of some JARID1 (Jumonji, AT-Rich Interactive Domain) family histone demethylases, as well as LSD1, via down-regulation of Sp1 expression [158]. Consistent with a decrease in the amount of the H3K4 demethylases, the tested HDAC inhibitor induced up-regulation of KLF4 and E-cadherin, two genes associated with tumor suppression and differentiation [158]. Thus, in addition to LSD1 the Jumonji (JMJ)-containing histone demethylases could represent valuable targets to inhibit in PCa. Nevertheless no JMJ inhibitor has been tested in PCa so far. Only some hydroxamic acids (Fig. 3), described as lysine demethylase (KDM)-4A/4C inhibitors, were tested in PCa cells and were found ineffective as single agents, although they displayed synergistic activity in combination with the tranylcypromine analogue NCL-2 [159].

Crea et al.

ACKNOWLEDGEMENTS FC contributed to manuscript writing and conception, and figure preparation. PLC contributed to manuscript writing and figure preparation. AM contributed to manuscript writing, figure preparation and critically revised the whole manuscript. CDH contributed to manuscript conception and writing and critically revised the manuscript. REFERENCES [1] [2] [3]

[4] [5] [6]

[7]

[8]

[9]

[10]

[11]

CONCLUSIONS A significant amount of pre-clinical data indicates that HPTMs are required for PCa initiation and progression. Those epigenetic marks are particularly important for a subset of stem-like PCa cells, which are responsible for the major clinical challenges in PCa (metastasis, drug resistance). Thus, the development of HPTMtargeting epi-drugs could pave the way to novel therapies. Most clinical studies have been focused on HDAC inhibitors, although histone acetylation seems not to play a prominent role in PCa CRCs. We expect that the development of effective PRC2 and possibly LSD1 inhibitors might be more effective, particularly for CRPCa. Before entering clinical trials, those molecules should be tested on appropriate pre-clinical models, in order to closely mirror the specific setting of application (e.g. adjuvant vs. palliative therapy). In addition, genetic and epigenetic profiling should be employed to identify signatures that predict drug efficacy/toxicity. Along with methylation, we think that PRC1-dependent histone ubiquitination could be another promising target. To the best of our knowledge, we are not aware of any molecule directly targeting BMI1. Nonetheless, we think that preclinical data indicate that BMI1 inhibitors may represent a further therapeutic opportunity for PCa CRC eradication. CONFLICT OF INTEREST All the authors declare no conflict of interest. This work was partially funded by grants to CDH from Prostate Cancer Canada (Grant #2010-556) and the Canadian Cancer Society (Grant #701097) (FC, PLC and CDH).

[12] [13]

[14] [15]

[16] [17] [18] [19] [20]

[21] [22] [23]

Shih C, Padhy LC, Murray M, Weinberg RA. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature 1981; 290(5803): 261-4. Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet 1993; 9(4): 138-41. Pettit SJ, Seymour K, O'Flaherty E, Kirby JA. Immune selection in neoplasia: towards a microevolutionary model of cancer development. Br J Cancer 2000; 82(12): 1900-6. Alexander S, Friedl P. Cancer invasion and resistance: interconnected processes of disease progression and therapy failure. Trends Mol Med.18(1): 13-26. Vecchione L, Jacobs B, Normanno N, Ciardiello F, Tejpar S. EGFR-targeted therapy. Exp Cell Res.317(19): 2765-71. Ebos JM, Kerbel RS. Antiangiogenic therapy: impact on invasion, disease progression, and metastasis. Nat Rev Clin Oncol.8(4): 21021. Sobrero A, Ackland S, Clarke S, et al. Phase IV study of bevacizumab in combination with infusional fluorouracil, leucovorin and irinotecan (FOLFIRI) in first-line metastatic colorectal cancer. Oncology 2009; 77(2): 113-9. Yau T, Wong H, Chan P, et al. Phase II study of bevacizumab and erlotinib in the treatment of advanced hepatocellular carcinoma patients with sorafenib-refractory disease. Invest New Drugs 2012; 30(6): 2384-90.. Tomblyn MB, Goldman BH, Thomas CR, Jr., et al. Cetuximab Plus Cisplatin, Irinotecan, and Thoracic Radiotherapy as Definitive Treatment for Locally Advanced, Unresectable Esophageal Cancer: A Phase-II Study of The SWOG (S0414). J Thorac Oncol 2012; 7(5): 906-12. Petrelli F, Borgonovo K, Cabiddu M, Ghilardi M, Barni S. Cetuximab and panitumumab in KRAS wild-type colorectal cancer: a meta-analysis. Int J Colorectal Dis.2011; 26(7): 823-33. Hensley ML. Big costs for little gain in ovarian cancer. J Clin Oncol 2011; 29(10): 1230-2. Taberlay PC, Jones PA. DNA methylation and cancer. Prog Drug Res 2011; 67: 1-23. Crea F, Nobili S, Paolicchi E, et al. Epigenetics and chemoresistance in colorectal cancer: an opportunity for treatment tailoring and novel therapeutic strategies. Drug Resist Updat 2011; 14(6): 280-96. Baylin SB, Ohm JE. Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat Rev Cancer 2006; 6(2): 107-16. Sunami E, de Maat M, Vu A, Turner RR, Hoon DS. LINE-1 hypomethylation during primary colon cancer progression. PLoS One 2011; 6(4): e18884. Mahapatra S, Klee EW, Young CY, et al. Global methylation profiling for risk prediction of prostate cancer. Clin Cancer Res.18(10): 2882-95. Crea F. Histone code, human growth and cancer. Oncotarget 2011; 3(1): 1-2. Kelly TK, De Carvalho DD, Jones PA. Epigenetic modifications as therapeutic targets. Nat Biotechnol 2011; 28(10): 1069-78. Dick JE. Looking ahead in cancer stem cell research. Nat Biotechnol 2009; 27(1): 44-6. Taylor RA, Toivanen R, Risbridger GP. Stem cells in prostate cancer: treating the root of the problem. Endocr Relat Cancer 2010; 17(4): R273-85. Lawson DA, Witte ON. Stem cells in prostate cancer initiation and progression. J Clin Invest 2007; 117(8): 2044-50. Crea F, Danesi R, Farrar WL. Cancer stem cell epigenetics and chemoresistance. Epigenomics 2009; 1(1): 63-79. Vincent A, Van Seuningen I. On the epigenetic origin of cancer stem cells. Biochim Biophys Acta 2012; 1826(1): 83-8.

Histone Modifications and Prostate Cancer [24]

[25]

[26]

[27]

[28] [29] [30]

[31] [32] [33] [34]

[35] [36]

[37]

[38] [39] [40] [41]

[42]

[43] [44] [45]

[46]

[47]

[48]

Bray F, Jemal A, Grey N, Ferlay J, Forman D. Global cancer transitions according to the Human Development Index (20082030): a population-based study. Lancet Oncol.2012; 13(8): 790801 Majumdar S, Buckles E, Estrada J, Koochekpour S. Aberrant DNA methylation and prostate cancer. Curr Genomics 2011; 12(7): 486505. Abdollah F, Schmitges J, Sun M, et al. Comparison of mortality outcomes after radical prostatectomy versus radiotherapy in patients with localized prostate cancer: A population-based analysis. Int J Urol 2012; 188(1): 73-83 Attard G, Reid AH, A'Hern R, et al. Selective inhibition of CYP17 with abiraterone acetate is highly active in the treatment of castration-resistant prostate cancer. J Clin Oncol 2009; 27(23): 3742-8. Hellerstedt BA, Pienta KJ. The current state of hormonal therapy for prostate cancer. CA Cancer J Clin 2002; 52(3): 154-79. Devlin HL, Mudryj M. Progression of prostate cancer: multiple pathways to androgen independence. Cancer Lett 2009; 274(2): 177-86. Beuzeboc P, Ropert S, Goldwasser F, Zerbib M. Management of metastatic castration-resistant prostate cancer following docetaxel. Bull Cancer. Bull Cancer 2012; 99 Suppl 1: S66-72. Bonkhoff H, Remberger K. Morphogenetic aspects of normal and abnormal prostatic growth. Pathol Res Pract 1995; 191(9): 833-5. Khan MO, Ather MH. Chromogranin A--serum marker for prostate cancer. J Pak Med Assoc 2011; 61(1): 108-11. Adolf K, Wagner L, Bergh A, et al. Secretagogin is a new neuroendocrine marker in the human prostate. Prostate 2007; 67(5): 472-84. Leong KG, Wang BE, Johnson L, Gao WQ. Generation of a prostate from a single adult stem cell. Nature 2008; 456(7223): 804-8. Richardson GD, Robson CN, Lang SH, Neal DE, Maitland NJ, Collins AT. CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci 2004; 117(Pt 16): 3539-45. Taylor RA, Toivanen R, Frydenberg M, et al. Human Epithelial Basal Cells Are Cells of Origin of Prostate Cancer, Independent of CD133 Status. Stem Cells 2012; 30(6): 1087-96. Goldstein AS, Lawson DA, Cheng D, Sun W, Garraway IP, Witte ON. Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics. Proc Natl Acad Sci USA 2008; 105(52): 20882-7. Signoretti S, Pires MM, Lindauer M, et al. p63 regulates commitment to the prostate cell lineage. Proc Natl Acad Sci USA 2005; 102(32): 11355-60. Marker PC. Does prostate cancer co-opt the developmental program? Differentiation 2008; 76(6): 736-44. Humphrey PA. Diagnosis of adenocarcinoma in prostate needle biopsy tissue. J Clin Pathol 2007; 60(1): 35-42. Wang X, Kruithof-de Julio M, Economides KD, et al. A luminal epithelial stem cell that is a cell of origin for prostate cancer. Nature 2009; 461(7263): 495-500. Ellwood-Yen K, Graeber TG, Wongvipat J, Iruela-Arispe ML, Zhang J, Matusik R, et al. Myc-driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 2003; 4(3): 223-38. Goldstein AS, Huang J, Guo C, Garraway IP, Witte ON. Identification of a cell of origin for human prostate cancer. Science 2010; 329(5991): 568-71. Visvader JE. Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis. Genes Dev 2009; 23(22): 2563-77. Wang S, Garcia AJ, Wu M, Lawson DA, Witte ON, Wu H. Pten deletion leads to the expansion of a prostatic stem/progenitor cell subpopulation and tumor initiation. Proc Natl Acad Sci USA 2006; 103(5): 1480-5. Cairns P, Okami K, Halachmi S, et al. Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res 1997; 57(22): 4997-5000. Whang YE, Wu X, Suzuki H, et al. Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression. Proc Natl Acad Sci USA 1998; 95(9): 5246-50. Komiya A, Suzuki H, Imamoto T, et al. Neuroendocrine differentiation in the progression of prostate cancer. Int J Urol 2009; 16(1): 37-44.

Current Pharmaceutical Design, 2014, Vol. 20, No. 00 [49]

[50]

[51]

[52] [53]

[54]

[55] [56]

[57]

[58] [59] [60]

[61] [62]

[63] [64]

[65] [66]

[67]

[68] [69] [70]

[71]

[72]

9

Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 2005; 65(23): 10946-51. Patrawala L, Calhoun T, Schneider-Broussard R, et al. Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene 2006; 25(12): 1696-708. Hurt EM, Kawasaki BT, Klarmann GJ, Thomas SB, Farrar WL. CD44+ CD24(-) prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis. Br J Cancer 2008; 98(4): 756-65. Duhagon MA, Hurt EM, Sotelo-Silveira JR, Zhang X, Farrar WL. Genomic profiling of tumor initiating prostatospheres. BMC Genomics.2011; 11: 324. Hurt EM, Chan K, Serrat MA, Thomas SB, Veenstra TD, Farrar WL. Identification of vitronectin as an extrinsic inducer of cancer stem cell differentiation and tumor formation. Stem Cells.28(3): 390-8. Kelly PN, Dakic A, Adams JM, Nutt SL, Strasser A. Tumor growth need not be driven by rare cancer stem cells. Science 2007; 317(5836): 337. Crea F, Mathews LA, Farrar WL, Hurt EM. Targeting prostate cancer stem cells. Anticancer Agents Med Chem 2009; 9(10): 1105-13. Klarmann GJ, Hurt EM, Mathews LA, et al. Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature. Clin Exp Metastasis 2009; 26(5): 433-46. Mulholland DJ, Kobayashi N, Ruscetti M, et al. Pten loss and RAS/MAPK activation cooperate to promote EMT and metastasis initiated from prostate cancer stem/progenitor cells. Cancer Res 2012.72(7): 1878-89. Kobayashi A, Okuda H, Xing F, et al. Bone morphogenetic protein 7 in dormancy and metastasis of prostate cancer stem-like cells in bone. J Exp Med.208(13): 2641-55. Visvader JE, Lindeman GJ. Cancer stem cells: current status and evolving complexities. Cell Stem Cell.2012; 10(6): 717-28. Qin J, Liu X, Laffin B, et al. The PSA(-/lo) Prostate Cancer Cell Population Harbors Self-Renewing Long-Term Tumor-Propagating Cells that Resist Castration. Cell Stem Cell 2012; 10(5): 556-69. Jeter CR, Liu B, Liu X, et al. NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene 2011; 30(36): 3833-45. Dubrovska A, Elliott J, Salamone RJ, et al. Combination therapy targeting both tumor-initiating and differentiated cell populations in prostate carcinoma. Clin Cancer Res 2010; 16(23): 5692-702. Cho YM, Kim YS, Kang MJ, Farrar WL, Hurt EM. Long-term recovery of irradiated prostate cancer increases cancer stem cells. Prostate.2012; 72(16): 1746-56. Glinsky GV. "Stemness" genomics law governs clinical behavior of human cancer: implications for decision making in disease management. J Clin Oncol 2008; 26(17): 2846-53. Glinsky GV. Stem cell origin of death-from-cancer phenotypes of human prostate and breast cancers. Stem Cell Rev 2007; 3(1): 7993. Ricci-Vitiani L, Pallini R, Biffoni M, et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 2010; 468(7325): 824-8. Mucci LA, Powolny A, Giovannucci E, et al. Prospective study of prostate tumor angiogenesis and cancer-specific mortality in the health professionals follow-up study. J Clin Oncol 2009; 27(33): 5627-33. Chen Z, Wang L, Wang Q, Li W. Histone modifications and chromatin organization in prostate cancer. Epigenomics 2010; 2(4): 551-60. Albany C, Alva AS, Aparicio AM, et al. Epigenetics in prostate cancer. Prostate Cancer.2011: 580318. Bjorkman M, Rantala J, Nees M, Kallioniemi O. Epigenetics of prostate cancer and the prospect of identification of novel drug targets by RNAi screening of epigenetic enzymes. Epigenomics 2010; .2(5): 683-9. Mathews LA, Crea F, Farrar WL. Epigenetic gene regulation in stem cells and correlation to cancer. Differentiation 2009; 78(1): 117. Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293(5532): 1074-80.

10 Current Pharmaceutical Design, 2014, Vol. 20, No. 00 [73]

[74]

[75]

[76]

[77] [78] [79] [80]

[81] [82] [83]

[84]

[85]

[86]

[87]

[88]

[89] [90] [91] [92]

[93] [94]

[95] [96]

Vogelmann J, Valeri A, Guillou E, Cuvier O, Nollmann M. Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation. Nucleus 2011; 2(5): 35869. Korolev N, Vorontsova OV, Nordenskiold L. Physicochemical analysis of electrostatic foundation for DNA-protein interactions in chromatin transformations. Prog Biophys Mol Biol 2007; 95(1-3): 23-49. Hodawadekar SC, Marmorstein R. Chemistry of acetyl transfer by histone modifying enzymes: structure, mechanism and implications for effector design. Oncogene 2007; 26(37): 5528-40. Chandrasekharan MB, Huang F, Sun ZW. Histone H2B ubiquitination and beyond: Regulation of nucleosome stability, chromatin dynamics and the trans-histone H3 methylation. Epigenetics.5(6): 460-8. Bycroft M. Recognition of non-methyl histone marks. Curr Opin Struct Biol.21(6): 761-6. Heightman TD. Chemical biology of lysine demethylases. Curr Chem Genomics 2011; 5(Suppl 1): 62-71. Wu JI. Diverse functions of ATP-dependent chromatin remodeling complexes in development and cancer. Acta Biochim Biophys Sin (Shanghai).44(1): 54-69. Allfrey VG, Faulkner R, Mirsky AE. Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis. Proc Natl Acad Sci USA 1964; 51: 786-94. Marmorstein R. Structure and function of histone acetyltransferases. Cell Mol Life Sci 2001; 58(5-6): 693-703. Kuo MH, Allis CD. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 1998; 20(8): 615-26. Ianculescu I, Wu DY, Siegmund KD, Stallcup MR. Selective roles for cAMP response element-binding protein binding protein and p300 protein as coregulators for androgen-regulated gene expression in advanced prostate cancer cells. J Biol Chem 2012; 287(6): 4000-13. Cang S, Feng J, Konno S, et al. Deficient histone acetylation and excessive deacetylase activity as epigenomic marks of prostate cancer cells. Int J Oncol 2009; 35(6): 1417-22. Chen H, Tu SW, Hsieh JT. Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer. J Biol Chem 2005; 280(23): 2243744. Bradley D, Rathkopf D, Dunn R, et al. Vorinostat in advanced prostate cancer patients progressing on prior chemotherapy (National Cancer Institute Trial 6862): trial results and interleukin6 analysis: a study by the Department of Defense Prostate Cancer Clinical Trial Consortium and University of Chicago Phase 2 Consortium. Cancer 2009; 115(23): 5541-9. Molife LR, Attard G, Fong PC, et al. Phase II, two-stage, singlearm trial of the histone deacetylase inhibitor (HDACi) romidepsin in metastatic castration-resistant prostate cancer (CRPC). Ann Oncol 2010; 21(1): 109-13. Schneider BJ, Kalemkerian GP, Bradley D, et al. Phase I study of vorinostat (suberoylanilide hydroxamic acid, NSC 701852) in combination with docetaxel in patients with advanced and relapsed solid malignancies. Invest New Drugs.2012; 30(1): 249-57. Schuettengruber B, Martinez AM, Iovino N, Cavalli G. Trithorax group proteins: switching genes on and keeping them active. Nat Rev Mol Cell Biol 2011; 12(12): 799-814. Shinkai Y. Regulation and function of H3K9 methylation. Subcell Biochem 2007; 41: 337-50. Richly H, Aloia L, Di Croce L. Roles of the Polycomb group proteins in stem cells and cancer. Cell Death Dis.2011; 2: e204. Morey L, Pascual G, Cozzuto L, et al. Nonoverlapping functions of the Polycomb group Cbx family of proteins in embryonic stem cells. Cell Stem Cell 2012; 10(1): 47-62. Tavares L, Dimitrova E, Oxley D, et al. RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3. Cell.148(4): 664-78. Hashimoto H, Vertino PM, Cheng X. Molecular coupling of DNA methylation and histone methylation. Epigenomics 2010; 2(5): 65769. van der Vlag J, Otte AP. Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation. Nat Genet 1999; 23(4): 474-8. Gould A. Functions of mammalian Polycomb group and trithorax group related genes. Curr Opin Genet Dev 1997; 7(4): 488-94.

Crea et al. [97]

[98]

[99] [100]

[101] [102]

[103]

[104] [105]

[106]

[107] [108]

[109] [110] [111]

[112]

[113]

[114]

[115]

[116] [117]

[118] [119]

Bernstein BE, Mikkelsen TS, Xie X, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006; 125(2): 315-26. Erhardt S, Su IH, Schneider R, et al. Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development. Development 2003; 130(18): 4235-48. Voncken JW, Roelen BA, Roefs M, et al. Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell cycle inhibition. Proc Natl Acad Sci USA 2003; 100(5): 2468-73. Lukacs RU, Memarzadeh S, Wu H, Witte ON. Bmi-1 is a crucial regulator of prostate stem cell self-renewal and malignant transformation. Cell Stem Cell 2010; 7(6): 682-93. Zhao JC, Yu J, Runkle C, et al. Cooperation between Polycomb and androgen receptor during oncogenic transformation. Genome Res.22(2): 322-31. Yu J, Yu J, Rhodes DR, et al. A polycomb repression signature in metastatic prostate cancer predicts cancer outcome. Cancer Res 2007; 67(22): 10657-63. Tan J, Yang X, Zhuang L, et al. Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells. Genes Dev 2007; 21(9): 1050-63. Crea F, Hurt EM, Mathews LA, et al. Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer. Mol Cancer 2011; 10: 40. Tong ZT, Cai MY, Wang XG, et al. EZH2 supports nasopharyngeal carcinoma cell aggressiveness by forming a corepressor complex with HDAC1/HDAC2 and Snail to inhibit Ecadherin. Oncogene 2012; 31(5): 583-94. Aguilo F, Zhou MM, Walsh MJ. Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARF-INK4a expression. Cancer Res 2011; 71(16): 5365-9. Ellinger J, Kahl P, von der Gathen J, et al. Global histone H3K27 methylation levels are different in localized and metastatic prostate cancer. Cancer Invest 2011; 30(2): 92-7. Shin YJ, Kim JH. The role of EZH2 in the regulation of the activity of matrix metalloproteinases in prostate cancer cells. PLoS One.2012; 7(1): e30393. Lu C, Han HD, Mangala LS, et al. Regulation of tumor angiogenesis by EZH2. Cancer Cell 2011; 18(2): 185-97. Vire E, Brenner C, Deplus R, et al. The Polycomb group protein EZH2 directly controls DNA methylation. Nature 2006; 439(7078): 871-4. Gravina GL, Marampon F, Piccolella M, et al. Hormonal therapy promotes hormone-resistant phenotype by increasing DNMT activity and expression in prostate cancer models. Endocrinology 2011; 152(12): 4550-61. Gal-Yam EN, Egger G, Iniguez L, et al. Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line. Proc Natl Acad Sci USA 2008; 105(35): 12979-84. Kunderfranco P, Mello-Grand M, Cangemi R, et al. ETS transcription factors control transcription of EZH2 and epigenetic silencing of the tumor suppressor gene Nkx3.1 in prostate cancer. PLoS One.2010; 5(5): e10547. Barwick BG, Abramovitz M, Kodani M, et al. Prostate cancer genes associated with TMPRSS2-ERG gene fusion and prognostic of biochemical recurrence in multiple cohorts. Br J Cancer.102(3): 570-6. Chen S, Bohrer LR, Rai AN, et al. Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2. Nat Cell Biol.12(11): 1108-14. Cha TL, Zhou BP, Xia W, et al. Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3. Science 2005; 310(5746): 306-10. Cao P, Deng Z, Wan M, et al. MicroRNA-101 negatively regulates Ezh2 and its expression is modulated by androgen receptor and HIF-1alpha/HIF-1beta. Mol Cancer 2010; 9: 108. Lu J, He ML, Wang L, et al. MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res 2011; 71(1): 225-33. Kong D, Heath E, Chen W, et al. Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM. PLoS One. 2102; 7(3): e33729.

Histone Modifications and Prostate Cancer [120]

[121] [122]

[123] [124] [125]

[126] [127]

[128]

[129] [130] [131]

[132] [133]

[134]

[135]

[136] [137]

[138]

[139] [140]

[141]


Schoeftner S, Sengupta AK, Kubicek S, et al. Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing. Embo J 2006; 25(13): 3110-22. Eskeland R, Leeb M, Grimes GR, et al. Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination. Mol Cell 201; 38(3): 452-64. Yu M, Mazor T, Huang H, et al. Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors. Mol Cell 2012; 45(3): 330-43. Akala OO, Clarke MF. Hematopoietic stem cell self-renewal. Curr Opin Genet Dev 2006; 16(5): 496-501. Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 2003; 423(6937): 255-60. Chen H, Zhou L, Dou T, Wan G, Tang H, Tian J. BMI1'S maintenance of the proliferative capacity of laryngeal cancer stem cells. Head Neck 2011; 33(8): 1115-25. Shimono Y, Zabala M, Cho RW, et al. Downregulation of miRNA200c links breast cancer stem cells with normal stem cells. Cell 2009; 138(3): 592-603. Zhang L, Jiao M, Li L, et al. Tumorspheres derived from prostate cancer cells possess chemoresistant and cancer stem cell properties. J Cancer Res Clin Oncol.2011; 138(4): 675-86. Crea F, Duhagon Serrat MA, Hurt EM, Thomas SB, Danesi R, Farrar WL. BMI1 silencing enhances docetaxel activity and impairs antioxidant response in prostate cancer. Int J Cancer 2011; 128(8): 1946-54. Fan C, He L, Kapoor A, et al. Bmi1 promotes prostate tumorigenesis via inhibiting p16(INK4A) and p14(ARF) expression. Biochim Biophys Acta 2008; 1782(11): 642-8. Fan C, He L, Kapoor A, et al. PTEN inhibits BMI1 function independently of its phosphatase activity. Mol Cancer 2009; 8: 98. Gu G, Yuan J, Wills M, Kasper S. Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo. Cancer Res 2007; 67(10): 4807-15. Varier RA, Timmers HT. Histone lysine methylation and demethylation pathways in cancer. Biochim Biophys Acta 2011; 1815(1): 75-89. Kahl P, Gullotti L, Heukamp LC, et al. Androgen receptor coactivators lysine-specific histone demethylase 1 and four and a half LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Res 2006; 66(23): 11341-7. Shi Y, Lan F, Matson C, et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 2004; 119(7): 94153. Amente S, Bertoni A, Morano A, Lania L, Avvedimento EV, Majello B. LSD1-mediated demethylation of histone H3 lysine 4 triggers Myc-induced transcription. Oncogene 2010; 29(25): 3691702. Shi YJ, Matson C, Lan F, Iwase S, Baba T, Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors. Mol Cell 2005; 19(6): 857-64. Thillainadesan G, Isovic M, Loney E, Andrews J, Tini M, Torchia J. Genome analysis identifies the p15ink4b tumor suppressor as a direct target of the ZNF217/CoREST complex. Mol Cell Biol 2008; 28(19): 6066-77. Metzger E, Wissmann M, Yin N, et al. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 2005; 437(7057): 436-9. Wang J, Lu F, Ren Q, et al. Novel histone demethylase LSD1 inhibitors selectively target cancer cells with pluripotent stem cell properties. Cancer Res 2011; 71(23): 7238-49. Chinnaiyan P, Vallabhaneni G, Armstrong E, Huang SM, Harari PM. Modulation of radiation response by histone deacetylase inhibition. Int J Radiat Oncol Biol Phys 2005; 62(1): 223-9. Sonnemann J, Bumbul B, Beck JF. Synergistic activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid and

Received: April 20, 2013

Accepted: July 18, 2013

[142]

[143]

[144] [145]

[146]

[147] [148]

[149]

[150]

[151]

[152]

[153]

[154] [155]

[156] [157]

[158]

[159]

11

the bisphosphonate zoledronic acid against prostate cancer cells in vitro. Mol Cancer Ther 2007; 6(11): 2976-84. Marrocco DL, Tilley WD, Bianco-Miotto T, et al. Suberoylanilide hydroxamic acid (vorinostat) represses androgen receptor expression and acts synergistically with an androgen receptor antagonist to inhibit prostate cancer cell proliferation. Mol Cancer Ther 2007; 6(1): 51-60. VanOosten RL, Earel JK, Jr., Griffith TS. Histone deacetylase inhibitors enhance Ad5-TRAIL killing of TRAIL-resistant prostate tumor cells through increased caspase-2 activity. Apoptosis 2007; 12(3): 561-71. Suenaga M, Soda H, Oka M, et al. Histone deacetylase inhibitors suppress telomerase reverse transcriptase mRNA expression in prostate cancer cells. Int J Cancer 2002; 97(5): 621-5. Kuefer R, Hofer MD, Altug V, et al. Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer. Br J Cancer 2004; 90(2): 535-41. Kim J, Park H, Im JY, Choi WS, Kim HS. Sodium butyrate regulates androgen receptor expression and cell cycle arrest in human prostate cancer cells. Anticancer Res 2007; 27(5A): 328592. Iacopino F, Urbano R, Graziani G, Muzi A, Navarra P, Sica G. Valproic acid activity in androgen-sensitive and -insensitive human prostate cancer cells. Int J Oncol 2008; 32(6): 1293-303. Chou YW, Chaturvedi NK, Ouyang S, et al. Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen responsiveness of prostate cancer cells. Cancer Lett 2011; .311(2): 177-86. Angelucci A, Muzi P, Cristiano L, et al. Neuroendocrine transdifferentiation induced by VPA is mediated by PPARgamma activation and confers resistance to antiblastic therapy in prostate carcinoma. Prostate 2008; 68(6): 588-98. Gravina GL, Marampon F, Giusti I, et al. Differential effects of PXD101 (belinostat) on androgen-dependent and androgenindependent prostate cancer models. Int J Oncol 2011; 40(3): 71120. Crea F, Paolicchi E, Marquez VE, Danesi R. Polycomb genes and cancer: Time for clinical application? Crit Rev Oncol Hematol 2012; 83(2): 184-93 Birney E, Stamatoyannopoulos JA, Dutta A, Guigo R, Gingeras TR, Margulies EH, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 2007; 447(7146): 799-816. Epub 2007/06/16. Shu L, Khor TO, Lee JH, et al. Epigenetic CpG demethylation of the promoter and reactivation of the expression of Neurog1 by curcumin in prostate LNCaP cells. Aaps J 2011; 13(4): 606-14. 13(4): 606-14. Forneris F, Binda C, Vanoni MA, Battaglioli E, Mattevi A. Human histone demethylase LSD1 reads the histone code. J Biol Chem 2005; 280(50): 41360-5. Lee MG, Wynder C, Schmidt DM, McCafferty DG, Shiekhattar R. Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol 2006; 13(6): 563-7. Ueda R, Suzuki T, Mino K, et al. Identification of cell-active lysine specific demethylase 1-selective inhibitors. J Am Chem Soc 2009; 131(48): 17536-7. Willmann D, Lim S, Wetzel S, et al. Impairment of prostate cancer cell growth by a selective and reversible lysine-specific demethylase 1 inhibitor. Int J Cancer 2012; 131(11): 2704-9 Huang PH, Chen CH, Chou CC, et al. Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases. Mol Pharmacol 2011; 79(1): 197-206. Hamada S, Suzuki T, Mino K, et al. Design, synthesis, enzymeinhibitory activity, and effect on human cancer cells of a novel series of jumonji domain-containing protein 2 histone demethylase inhibitors. J Med Chem 2010; 53(15): 5629-38.