The role of epigenetic regulation in stem cell and cancer biology

J Mol Med (2012) 90:791–801 DOI 10.1007/s00109-012-0917-9

REVIEW

The role of epigenetic regulation in stem cell and cancer biology Lilian E. van Vlerken & Elaine M. Hurt & Robert E. Hollingsworth

Received: 27 February 2012 / Revised: 4 May 2012 / Accepted: 10 May 2012 / Published online: 2 June 2012 # Springer-Verlag 2012

Abstract Normal development and homeostasis requires a carefully coordinated gene expression program. Appropriate transcriptional regulation is maintained, in part, through epigenetic modifications of both DNA and histones. It is now apparent that the epigenetic landscape is complex and carefully controlled to both silence and activate gene transcription and that these states remain exquisitely poised for reversal. The deregulation of epigenetics in cancer is common and results in both the activation of oncogenes and the silencing of tumor suppressors. A tremendous amount of research corroborates the existence in many tumor types of a cancer stem cell that is both the origin and cell type responsible for resistance of tumors to current therapies. As our understanding of cancer stem cell biology continues, it is apparent that these cells are also under the influence of epigenetic regulation. We will discuss the cancer stem cell hypothesis and the role of epigenetics in both normal and cancer stem cell biology. Keywords Cancer stem cells . Polycomb repressive complex . Histone modifications . DNA methylation

Introduction In the late nineteenth century, the German pathologist, Rudolf Virchow, postulated that all cells including cancerous cells arise from other primitive cells [1]. However, it wasn’t until a century later, that Virchow’s idea was tested L. E. van Vlerken : E. M. Hurt : R. E. Hollingsworth (*) Oncology Research, MedImmune, LLC, One MedImmune Way, Gaithersburg, MD 20878, USA e-mail: [email protected]

by dissecting the hierarchy of leukemia cells [2]. Dick and colleagues showed acute myeloid leukemia (AML) originates from a primitive cell type with stem cell-like properties [3]. Among a mixture of different leukemic cell types, these rare cancer stem cells (CSCs) reinitiated the disease when serially transplanted. This and related work suggested a hierarchical model for cancer formation which purports that only a small proportion of tumor cells are tumorigenic, rather than arising by a stochastic etiology in which all tumor cells have a similar but low tumorigenic capacity ([4]; Fig. 1a). Several mechanisms may be responsible for the genesis of CSCs, each of which involves reactivation of “stemness” genes responsible for the behavior of these cells (Fig. 1b). The primary defining characteristics of CSCs are their ability to self-renew and reinitiate cancer with a heterogeneous cellular morphology that is indistinguishable from the original tumor [5]. This is the basis of CSC identification experiments in which subsets of cancer cell types defined by unique cell surface markers are serially transplanted in immunocompromised mice to test for their ability to reform the tumor. Following the identification of AML CSCs, they have been identified in other hematologic malignancies [6] and many solid tumors, including breast [7], brain [8], prostate [9], lung [10], liver [11], colon [12], pancreas [13], ovary [14], and head and neck [15]. However, even within a single cancer type, several markers have been used to define a CSC population by different investigators and the best marker profile to elucidate CSCs often remains controversial (for example, see [16] for colorectal cancer). Alternatively, it could be that several CSC states exist [3, 17, 18], each with their own marker profile, or that CSC phenotypes can vary from patient to patient [19]. Even despite this complexity, CSCs may still be functionally defined as the tumorigenic component within tumors and may have a

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Fig. 1 Model for and possible origins of cancer stem cells. a The stochastic and CSC models of tumorigenesis. (Adapted from [4]). The stochastic model asserts that every tumor cell has a low but equal probability of proliferating limitlessly and thus the potential to behave as a stem cell. In contrast, the CSC model proposes that only a distinct subset of cells will consistently have the capacity to initiate tumor growth and reproduce the hierarchy of cell types that comprise the tumor. Recent research supports the CSC model for many cancer types, including experiments showing that CSCs isolated from tumors are the only population capable of forming tumor when injected in low numbers into mice. b CSC may arise from several sources. The similarity between some CSC populations and normal stem cells suggests that the

former arises from the latter, with additional mutations driving a cancerous state. The long life span of stem cells relative to mature, differentiated cells has been used to suggest that these cells are more likely to accumulate the multiple genetic mutations necessary for tumor formation. CSCs also may arise by mutation of a progenitor cell within a cellular hierarchy. Alternatively, dedifferentiation of malignant cells to a stem cell-like state may occur through mutation. Regardless of their origin, CSCs possess several properties that distinguish them from the majority of cells in a tumor. These include activation of stemness gene expression programs by transcriptional and epigenetic regulatory machinery

common genomic signature despite differing cell surface phenotypes [19]. While we are still uncovering the complexities of CSC biology, several general properties have been described. In the laboratory, CSCs from some cancers are resistant to radiation [21–26] and some chemotherapy drugs [20, 27–30] that effectively kill other cancer cells. This provides

one explanation for the frequent relapse seen in patients following current treatments. Although this hypothesis has yet to be fully confirmed using a CSC-directed therapy in cancer patients, several studies have correlated CSCs with poor prognosis [31, 32]. For example, glioblastoma multiforme (GBM) patients whose tumors bear a relatively high proportion of CSCs (CD133+ cells), suffer from decreased

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response to therapy, higher malignancy, and significantly lower survival time [31]. Some CSCs express high levels of ATP-binding cassette transporters [27, 33, 34], hyperactivate DNA repair mechanisms [35–37], and downregulate apoptotic pathways [26, 38]. Together with their quiescent state, these properties ostensibly explain their chemoresistance [26, 27]. As the cells that, for some cancers, appear to be responsible for tumor initiation and resistance to current treatments, CSCs are an important target for new drugs. Similarities to normal stem cell (SC) physiology have aided in the elucidation of CSC biology, but also compel the need to identify differences for selective targeting. Like normal SCs, CSCs can exist in a quiescent state, are longlived, and appear to have an indefinite proliferative potential [39–41]. They are capable of symmetric division (self-renewal) as well as asymmetric division (mulitpotency), spawning both a CSC and the more short-lived progeny comprising the tissue or tumor. They possess mesenchymal properties, including the ability to migrate [42–44] that for CSCs may be linked to tumor metastasis. SCs and CSCs are also highly influenced by signals in their microenvironment, and often reside in specialized niches within tissues [45]. Some CSCs express established SC markers and ‘stemness’ regulators, including NANOG, OCt3/4, and SOX2 [46]. Several SC signaling pathways that normally regulate embryogenesis and development are deregulated in CSCs. The most studied pathways are those regulated by Wnt, Notch, and Hedgehog [47]. These pathways regulate cell fate, proliferation, migration, and differentiation by controlling complex gene expression programs but are hyperactivated in several cancers [48]. SC signaling also involves epigenetic regulation in which DNA and chromatin remodeling lead to transcriptional program changes [49]. For example, the polycomb group (PcG) proteins have emerged recently as important players in maintaining SC multipotency [50–52]. In fact, several lines of evidence have suggested that epigenetic regulation of chromatin function is a common feature of SCs, including CSCs [53, 54]. These findings add a new layer of comprehension to the function of CSCs, and are the subject of this review.

Epigenetic regulation and cancer Modifications to both DNA and histones have a profound effect on transcription and are the best understood of epigenetic changes. While DNA methylation can silence transcription, a range of post-translational modifications of histones including methylation, acetylation, and ubiquitination, results in a dynamic regulation. These modifications and their overall effects on transcription will be discussed below.

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DNA methylation DNA is typically methylated on the 5′ position of cytosine (5mC) and, in mammals, methylation is initiated and maintained by three related enzymes, DNA methyltransferases (DNMT) 1, 3a, and 3b [55]. Genomic DNA is not uniformly methylated, but is rather hypermethylated in repetitive and foreign elements and hypomethylated at focused locations (CpG islands) within gene promoters. CpG islands are found in approximately 40 % of promoters in the mammalian genome [56] and exhibit an open chromatin formation, are associated with transcriptionally active genes, and are hotspots for histone modifications [57]. Methylation of cytosines within CpG islands typically results in transcriptional silencing (Fig. 2a), most dramatically seen in the developing embryo where methylation is responsible for X-inactivation and imprinting [58]. While the mediators of mammalian DNA methylation have been identified for decades, and the existence of demethylating enzymes has likewise been hypothesized, it was only recently that enzymes actively involved in demethylation have been identified. In 2009, Tahiliani et al. determined that ten-eleven-translocation (TET), a gene identified as a fusion partner of mixed-lineage leukemia (MLL) [59], was identified as an enzyme capable of converting 5mC to 5-hydroxymethylcytosine (5hmC) [60]. Overexpression of TET1 resulted in decreased 5mC levels, while knock-down decreased 5hmC levels by 40 %. The ability of 5hmC to be converted to unmodified cytosine has been reported in bacteria and suggests that 5mC, converted to 5hmC by TET, can be further actively demethylated in mammalian cells [61]. In addition to TET, activation induced cytidine deaminase (AID) can promote demethylation [62]. AID was previously shown to be required for 5mC demethylation in zebrafish by converting 5mC to thymine which is then repaired by the G/T mismatch specific thymine glycosylase, Mbd4 [63]. Like nearly all normal biologic processes, aberrant DNA methylation has been noted in cancer. While in healthy cells there is relative hypomethylation of CpG islands, in cancer there is an increase in de novo methylation resulting in hypermethylation of CpG islands [64]. This hypermethylation silences a diverse group of tumor suppressor genes, including genes involved in DNA repair, cell-cycle control, apoptosis, adhesion, and metastasis [65]. With the advent of methylation specific arrays, a wealth of knowledge is being gained about the role of DNA methylation in cancer. Genome wide patterns of methylation were recently determined in AML [66]. The authors confirmed the general association between DNA methylation and transcriptional silencing and identified CpG islands correlated with prognosis, proving that aberrant DNA methylation plays an important role in cancer progression and patient survival. Moreover, they found an association among altered methylation of polycomb target genes, AML progenitor cells, and

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Fig. 2 Schematic representation of epigenetic modifications. a Enzymes involved in DNA methylation (Me). DNMT1 is primarily responsible for maintenance of DNA methylation during replication while DNMT3a and 3b are primarily de novo methyltransferases. b Lysine and arginine residues found in the indicated histones (H2A, H2B, H3, and H4), where Me refers to methylation and Ac refers to acetylation. Modifications can result in either activation (green) or repression (red) of transcriptional activity

prognosis. Thus, linking aberrant DNA methylation and CSCs. In addition to gene silencing by direct promoter methylation, it is also well known that modification of chromatin structure can lead to regulation of gene expression. Chromatin describes DNA compaction in the nucleus, and is structurally made up of the nucleosome, 146 bp DNA units wrapped around two copies of each histone, H2A, H2B, H3, and H4 [67]. N-terminal tails of these histones can undergo a variety of post-translational modifications that ultimately determines the structure and accessibility of the chromatin regions [68]. This regulation of chromatin structure is termed the “histone code” [69]. Histone modifications epigenetically regulate gene expression by either changing the accessibility of chromatin to transcription mediators, or by recruiting effector proteins that read this histone code [69]. A variety of histone modifications have been discovered, including acetylation, methylation, ubiquitination, and phosphorylation, summarized in Fig. 2b [70]. Methylation and acetylation of histones are best characterized, as compared to the other modification, for their pathological role in cancer. Additionally, ubiquitination and phosphorylation appear to function as stepping stones to recruit effector modifications in methylation and acetylation, thus in the context of this review methylation and acetylation will be more extensively discussed. Histone methylation Lysine and arginine residues on histones are methylated through enzymatic action of histone methyltransferases [71]. Arginine can accept either one or two methyl groups

(di-methylation can be either symmetrical or asymmetrical) [72] while lysine can accept up to three methyl groups [71]. Reviews on histone arginine [72] and lysine [73] methylation further detail these events, and thoroughly discuss their relative roles in gene repression and activation. The three most notable methylation events are on H3K4, H3K27, and H3K9, whereby H3K4me 3 activates and H3K27me 3 and H3K9me 3 repress transcription [74]. H3K4 methylation is catalyzed by the trithorax group, specifically through activity of SET1 or MLL-family members [75, 76]. H3K27 and to some extent H3K9 are catalyzed by the PcG, through activity of EZH2 which resides in polycomb repressive complex (PRC)2 [75, 76]. However, H3K9 is predominantly mono- and di-methylated by G9a [77]. Interestingly, while H3K27 and H3K9 methylation are both repressive marks, they differ greatly in their protein interactions. H3K27me3 recruits PRC1 that ubiquitinates K119 of histoneH2A (H2AK119ub) through the action of Ring1b mediated by BMI1 [76, 78]. Methylation of H3K9, on the other hand, recruits HP1 which further interacts with DNMTs [79]. Methylation of histone arginine residues has not been implicated in directly activating or repressing genes yet. Rather, it appears that arginine methylation impacts binding of effector molecules [72]. Interestingly, many sites of arginine methylation are close in proximity to lysine methylation sites, such as the proximity of H3R2 to H3K4, H3R8 to H3K9, and H3R26 to H3K27. At H3R2/H3K4 pairs, cross-talk between arginine and lysine methylation has been defined [80]. H3R2 is predominantly methylated by PRMT6, an enzyme that can also methylate H3K4. However, tri-methylation of H3K4 by MLL, essential for gene activation, is inhibited if H3R2 is

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methylated. This suggests that arginine methylation may act as a switch, regulating important lysine methylation events. It is still unclear whether the H3R8/H3K9 and H3R26/H3K27 pairs function in a similar regulatory fashion, although the prospect is intriguing. While most oncology research centers on methylation at K4, K9, and K27, a variety of other histone methylation events are relevant as oncogenic mediators. Methylation at H3K36 acts to repress methylation activity at H3K27 [81], suggesting a mechanism whereby H3K36 methylation is associated with transcriptional activation. In addition, H3K79 methylation, which activates transcription, is important in leukemogenesis [82] and is essential for cell proliferation in lung cancer cells [83]. Similarly, trimethylation at H4K20 plays a role in recruitment of checkpoint proteins in DNA damage repair [84], thus the progressive loss of H4K20me3 in non-small cell lung cancer correlates with this tumor suppressive function of this methylation event [85]. Histone methylation is a dynamic process through its tight regulation of transcription. However, regulation is complicated by the discovery of histone demethylases (HDMs), further shattering the dogma that methylation is irreversible. HDMs belong to either of two classes of enzymes, the KDM1/LSD1 family, and the much larger JmjC domain-containing family [86]. KDM1 predominantly removes mono- and di-methylated H3K4 [87], but in the presence of nuclear steroid receptors, it can also remove mono- and di-methylated H3K9 [88]. Removal of monoand di- methylated H3K9 is in other instances catalyzed by JMJD1/KDM3 proteins [89]. Demethylation of trimethylated H3K4 and tri-methylated H3K9 are catalyzed predominantly by the JARID1/KDM5, and JMJD2/KDM4 classes, respectively, both classes of enzymes that belong to the JmjC domain containing family [90]. Demethylation of H3K27 is catalyzed by the KDM6 family of proteins, better known as UTX and JMJD3 [91]. The importance of histone demethylation as an oncogenic process is seen in expression patterns of these demethylase enzymes in tumors. KDM1/ LSD1 has been shown to be overexpressed (>2-fold) in cancers of the lung, bladder, colorectal, and leukemias [92]. Members of the JARID1/KDM5 family are similarly overexpressed (>2-fold) in a wider variety of tumor types [92], suggesting that repression of transcription through demethylation of H3K4 is an oncogenic phenomenon. Similarly, members of the JMJD2/KDM4 family are downregulated in tumor tissues, most notably in leukemias and lymphomas but also in breast and renal cancer [92]. This supports a mechanism whereby repressed domains can become active. Histone acetylation Histone acetylation is another epigenetic modification regulating transcription. Acetylation occurs on ε-amino groups of

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lysine residues, catalyzed by a large family of proteins termed histone acetyl transferases (HATs). Acetylation may slightly decrease the net positive charge on lysine, hindering the affinity of histones to negatively charged DNA, and facilitating the binding of transcriptional mediators [93]. Acetylation, thereby, is largely associated with transcriptional activation. Many lysine acetylation sites have been identified [93], thus we will limit discussion to a small subset of acetylation events that function in oncogenesis. One of the first characterized events is acetylation of H4K16 (H4K16ac). There is a global loss of H4K16ac in a variety of tumor types, such as lymphoma, breast, colon, and lung, compared with normal tissue [94]. Interestingly, the same samples showed a similar loss of H4K20me3, suggesting that these two events could be used as a signature in carcinogenesis. H3K56 acetylation, associated with apoptosis and DNA repair [95], is common in a variety of tumors, interestingly also correlated to a dedifferentiated state of cells [95]. Contrary, loss of acetylation at H3K18 is a prognostic marker in prostate, lung, and renal cancer [96]. While histone acetylation is generally associated with transcriptional activation, the genes regulated by these marks can be oncogenes or tumor suppressors. The more interesting lysine residues are the ones where both methylation and acetylation occur. A well-characterized example of this is the H3K9 site, where acetylation and methylation regulates a switch between activation and repression of genes [97]. As these marks regulate opposing events, it is postulated that acetylation at this site prohibits methylation. A similar methyl/acetyl switch was found for H3K27, where mice deficient in Suz12 had decreased H3K27me3 levels, replaced by increased H3K27ac levels [98]. As with histone methylation, histone acetylation is also reversible. In fact, of the histone modifications, deacetylation is perhaps most widely recognized as an oncogenic mediator. Deacetylation is catalyzed by histone deacetylases (HDACs), and inhibitors against HDACs were first FDA approved for cancer treatment earlier this decade. However the oncogenic function of HDACs is not specified by its deacetylase activity on histones only. HDACs also play a role as transcriptional cofactors, where they interact directly with transcription factors such as E2f, Stat3, p53, Rb, NF-κB, and others [99]. Moreover, they deacetylate non-histone proteins influencing other processes upstream of chromatin remodeling [100]. A more detailed discussion on HDAC inhibitors, in CSC therapy specifically, follows in “The emerging role of epigenetic regulation in CSCs and possible therapeutic intervention.” HDACs counter HATs by tightening the interaction between DNA and histones, thereby diminishing access for transcription factors. Although this is entirely a repressive function, depending on the target genes, this repression can be either tumor promoting or suppressive. The class I enzymes HDAC1 and HDAC2, suggested to play compensatory functions in development [101], have both been shown

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to be upregulated in a wide variety of tumor tissues [92]. However, SIRT1 and SIRT2, belonging to class III HDACs, were largely downregulated in tumor tissues over normal [92].

The role of epigenetics in maintaining a SC state Epigenetic regulation, in the form of DNA methylation and histone modification (schematically depicted in Fig. 3), plays a key role in the transcriptional program of normal SCs, thus we can use clues from this regulation to deduce the mechanisms that might be prevalent in CSCs as well. DNA methylation Recently the role de-novo methylation in the regulation of SC differentiation has been recognized. Challen and colleagues showed that serial transplantation of hematopoietic SCs (HSCs) from Dnmt3a-null mice [102] resulted in a 200fold expansion in HSCs but whose differentiation capacity decreased with each passage. They also found a reduction in the methylation of SC-specific genes that could provide a possible explanation for the observed expansion of HSCs. This led the authors to conclude that DNMT3a is required

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for proper differentiation of HSCs and shows a mechanistic link between SC maintenance and DNA methylation. Pluripotency of ESCs makes them a great tool to study the epigenetic changes regulating self-renewal and differentiation, two opposing programs. Interestingly, studies have found that crucial pluripotency factors, such as NANOG, OCT3/4, and SOX2 are regulated through epigenetics. The Nanog promoter in germ cells was found to be hypomethylated in correlation with differentiation of these cells [103]. Furthermore, several key pluripotency factors, such as Nanog and Sox2, are themselves regulated through OCT4-dependent transcription [104, 105]. And Oct4, in turn, is also epigenetically repressed, regulated by Cdx2 and Brg1, although independent of DNA methylation [106]. Several groups, including our own, have found upregulation of several pluripotency factors in CSCs [107–110]. Many lessons regarding the importance of epigenetics in maintaining stemness can be taken from iPS cells. In 2006, Takahashi and Yamanaka showed that a limited set of transcription factors, Oct3/4, Sox2, Klf4, and Myc, could reprogram adult somatic cells into pluripotent cells [111]. Mikkelson et al. recapitulated this work and further built upon this to show that the chromatin maps of these iPS cells was strikingly similar to the open chromatin structure of ESCs, with a strong presence of bivalent (H3K27me/H3K4me) chromatin and frequent DNA hypomethylation [112]. Interestingly, inhibition of DNMT1 facilitated reprogramming [112]. It was further shown that AID, the DNA demethylase, can demethylate both NANOG and OCT4 [62], and important step in reprogramming. These results underscore the importance of epigenetic regulation in pluripotency. Histone modifications Bivalent marking on chromatin, whereby areas marked by H3K27 methylation are found alongside areas marked by H3K4 methylation [113], regulates chromatin for repression (H3K27 methylation), while simultaneously keeping it poised for transcriptional activation (H3K4 methylation). By these means, the genome remains flexible to produce progeny that either retains pluripotency, or becomes more lineage committed. Several developmental regulators are regulated in this manner [114]. Activation of these developmental regulators, such as Hox-, Fox-, Sox-, and Gata-family transcription factors, promotes differentiation, thus silencing in ESCs would aide in retention of pluripotency [115].

Fig. 3 Epigenetic modifications result in altered transcriptional activity. As histones are modified there is a change from active transcription to silencing. The key events are denoted in this simplified schematic diagram. Methylation of H3K4, as well as acetylation of histones, accompany an active transcriptional state while methylation of H3K9 and H3K27 are associated with inactive DNA. Moreover, DNMTs can methylate DNA at CpG islands and silence loci further

The emerging role of epigenetic regulation in CSCs and possible therapeutic intervention Perhaps the best characterized occurrence of epigenetic regulation in CSCs is through PcG gene silencing. We have

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found that isolated CSCs of breast and pancreatic cancer overexpress EZH2, the PRC2 protein responsible for trimethylating H3K27, and that EZH2 was essential for maintenance of an intact CSC population (unpublished results). Similar reports demonstrated an importance for EZH2 in CSCs of prostate [54], ovarian [116], and GBM [117]. PRC1 acts downstream of PRC2 to recognize the repressive H3K27me3 mark and inhibit further transcription sterically or by ubiquitinating H2AK119 [76]. Interestingly while Ring1b is the catalytic component of PRC1, it is most often BMI1 a PRC1 co-repressor to Ring1b that is correlated to cancer and CSCs. Facchino et al. found that BMI1 can help confer radioresistance to neural cancer SCs by promoting DNA damage repair [118], suggesting a role for epigenetic regulation not only in tumorigenesis of CSCs, but also therapeutic escape. Yang et al. uncovered the necessity for BMI1 in tumor initiation and EMT (a process closely linked to CSCs [119]) in HNSCC [120]. BMI1 is overexpressed in CSCs of GBM, and directly linked to tumorigenicity of this cancer [121]. So if H3K27me3 plays a role in CSCs, what happens to H3K4me3? Inhibitors of LSD1, the HDM responsible for demethylating H3K4, specifically acted on pluripotent cancer cell types such as teratocarcinomas, seminomas, and embryonic carcinomas [122], suggesting a downregulation of H3K4 methylation in tumorigenicity of these cell types. However, it is unclear whether these results would extend to CSCs of more differentiated tissue types. The inhibition of histone modifications for targeting CSCs is being modeled pre-clinially. In particular, several studies have incorporated the use of 3-deazaneplanocin A (DZNep), a methyltransferase inhibitor shown to disrupt the PRC2 complex [123]. These studies demonstrate that DZNep can reduce CSCs in AML [124], HCC [125], GBM [126], and prostate [54]. However, the clinical usefulness of this drug is questionable due to specificity [127] and pharmacokinetics [128] as well as largely unexplored toxicity [129]. It is also important to note that epigenetic mechanisms can operate differently amongst diverse tumor types. For example, while EZH2 is largely shown to be oncogenic,

a recent study by Simon et al. found that genetic disruption of EZH2 actually promoted the onset of mouse T cell acute leukemia, thereby suggesting a tumor suppressive role for EZH2 in at least this tumor type [130]. Therefore, a greater clarity of the role that EZH2 specifically, and epigenetics in general, plays in diverse settings is fundamental. While there is encouraging pre-clinical data suggesting that targeting PRCs may be effective against CSCs, we will need to wait until these reach the clinic to determine their true usefulness and benefit to cancer patients. Specific acetylation events have not been pinpointed in CSCs as of yet, however, the occurrence of histone acetylation in CSCs and tumorigenesis can be implied from the effect of HDAC inhibitors in tumors. Nalls et al. found that miR-34a was frequently downregulated in pancreatic CSCs, thought to occur through epigenetic silencing. Treatment with the HDAC inhibitor Vorinostat, or with the demethylating agent 5-Aza-dC, separately restored miR-34a expression in pancreatic CSCs, which led to decreased viability and EMT inhibition in pancreatic CSCs [131]. These data support the idea that in addition to histone methylation, histone acetylation can also play an important role in regulating CSCs. Epigenetic silencing through DNA methylation is also a means of regulating CSCs. Many reports have shown that CD133, a CSC marker in colon, ovarian, HCC, prostate, and brain, is directly regulated through promoter methylation [132–135]. Furthermore, in colon cancer it was found that DNA methylation of Wnt-target genes, ASCL2 and LGR5, was associated with tumorigenesis [136]. Earlier studies implicated these two factors in colon CSCs [137]. Hypomethylating agents, such as azacitidine and decitabine (5-aza-2′-dioxycytidine), are cytosine analogs that are incorporated into DNA and bind irreversibly with DNMT1. Both azacitidine and decitabine have been used in numerous preclinical studies showing that they can reverse methylation, re-express previously silenced genes and lead to death of cancer cells (For a thorough review see [138]). The general association of DNA hypermethylation and genes involved in differentiation of SCs [139], suggests that the

Table 1 The role of epigenetic modification in CSCs Target

Phenotype

Tumor type

Reference

Histone methylation (H3K27me3) DNA hypomethylation

EZH2

LSD1 HDAC

Breast, pancreatic, prostate, ovarian, and glioblastoma Ovarian, colon, HCC, prostate, and glioblastoma Teratocarcinoma, seminoma, and embryonic carcinoma Pancreatic

[54, 116–117]

Histone methylation (H3K4me3) Histone acetylation

[131]

DNA hypermethylation

LGR5 and ASCL2

Manipulation of EZH2 directly influences CSC frequency Directly regulates expression of CD133, a CSC marker Inhibition of LSD1 specifically targets pluripotent carcinomas Treatment with HDACi 5-Aza-dC inhibited CSC viability and EMT Epigenetic silencing of LGR5 and ASCL2 reduced tumorigenesis

Colon

[136]

CD133

[132–135] [122]

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use of hypomethylating agents have the potential to induce differentiation of CSCs thereby sensitizing them to many therapies effective at eliminating the bulk of tumor cells.

Conclusions Our understanding of the exact role epigenetic regulation plays in CSC biology is just beginning (Table 1). Evidence is linking epigenetic regulation directly to pluripotency transcription factors and self-renewal pathways, known to play a critical role in the maintenance of CSCs. This knowledge leads not only to a better understanding of how the cell of origin may arise in cancer, but also helps identify root causes to target and help eradicate CSCs. Promise of this strategy is seen in studies where therapeutic intervention with HDAC and PRC inhibitors directly affects CSCs. Continued exploration into epigenetic regulation of CSCs may provide critical insights into the fundamental means of CSC regulation and may open the path for novel therapeutic opportunities.

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