Epigenetic Regulation/Dysregulation in Cancer Stem Cells: Comparison
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Please note this is a comparison between Version 2 by Dean Liu and Version 1 by Cristabelle De Souza.

EIn cancer, several of pigeneticost-translational modifiers of Cancer Stem Cell survivacations can undergo dysregulation, driving intratumoral heterogeneity and leading to tumor subpopulations with novel epigenetic regulation. These epigenetic regulations are carried out mainly by histone writers, erasers and readers.

  • cancer stem cells
  • epigenetics
  • tumoral plasticity

1. Introduction

 Histones are proteins that are involved in the regulation of gene expression and silencing through interaction with DNA. For example, H3K9 trimethylation (addition of three methyl groups to lysine residue at position 9 on histone 3) together with deacetylation of histones H4 and H3 is associated with gene repression, while H4K8 (lysine residue at position 8 of histone 4) acetylation and H3K14 (lysine residue at position 14 of histone 3) acetylation along with H3S10 (serine residue at position 10 of histone 3) phosphorylation is associated with gene expression [26][1]. In cancer, several of these post-translational modifications can undergo dysregulation, driving intratumoral heterogeneity and leading to tumor subpopulations with novel epigenetic regulation. These epigenetic regulations are carried out mainly by histone writers, erasers and readers.

2. Classification of Epigenetic Mediators: Writers, Erasers and Readers

2.1. Writer Enzymes

Writers are enzymes that are involved in adding methyl or acetyl groups to specific amino acid residues of histones or methyl group to cytosine nucleotides of DNA [25][2]. Adding methyl groups to CpG islands present in DNA or histone tails helps prevent transcription by blocking transcription factors [25][2]. Transcription can be further prevented by the recruitment of proteins that bind to methylated sites, creating additional inaccessible binding sites on the chromatin [25][2]. DNA methyltransferases (DNMT) are writer enzymes that are involved in adding a methyl group to cytosine residues that are a part of CpG dinucleotides, forming 5-methylcytosine [27][3]. CpG dinucleotides are found in CpG islands in promoters [27][3]. Methylation of these regions in genes is associated with gene silencing [27][3]. DNMTs are divided into DNMT1 and de novo DNMT. De novo DNMTs are expressed largely during development and are important in the maintenance of methylation patterns in human embryonic stem cells, whereas during differentiation DNMT1 becomes highly expressed, with a reduction in de novo DNMTs [27][3]. DNMT1s are responsible for ensuring proper inheritance of epigenetic patterns during replication [27][3]. Several studies have shown the importance of DNMT1 in the maintenance of the CSC phenotype [28][4]. For example, a knockout of DNMT1 led to a reduction of cancer stem cell markers such as high expression levels of ALDH (aldehyde dehydrogenase), CD44+ and CD24+ in colon cancer cell lines [29][5]. Histone lysine methyltransferases (KMTs) are also a type of writer enzyme involved in the transfer of one, two or three methyl groups to specific lysine (K) positions at the tails of histones, which correspond to different biological responses [27][3]. Methylation at positions H3K4 (lysine residue at position 4 of histone 3), H3K35 (lysine residue at position 35 of histone 3) and H3K79 (lysine residue at position 79 of histone 3) is associated with gene expression, or open chromatin, while methylation at positions H3K27 (lysine residue at position 27 of histone 3), H3K9 (lysine residue at position 9 of histone 3) and H4K20 (lysine residue at position 20 of histone 4) is associated with gene repression [27][3]. A trimethylation pattern has been associated with transcriptionally active promoters, while monomethylation has been associated with active enhancers [30][6]. KMTs (lysine methyltransferases) can be subdivided based on the presence of the SET [Su(var)3–9 Enhancer-of-zeste and Trithorax] domain [30][6]. These enzymes are highly site-specific and are responsible for adding mono, di and tri methyl groups to histones [30][6]. A list of these enzymes is well detailed in C. Hon and Hawkins [30][6]. Several of the KMT enzymes are over-expressed in multiple cancers [27][3]. Enhancer of Zeste-Homolog 2 (EZH2), Enhancer of Zeste-Homolog 3 (EZH3) and H3K27(lysine residue at position 27 of histone 3) KMTs were found at elevated levels in glioma, breast and leukemia CSCs, and are credited with maintaining their quiescent state [31][7].

2.2. Eraser Enzymes

Erasers remove epigenetic modifications from DNA and histones. Histone demethylases remove methyl groups from histones; KDMs (histone lysine demethylases) remove methyl groups from lysine specifically. Lysine-specific demethylases are categorized into two groups: the FAD (flavin adenine dinucleotide)-dependent KDMs, KDM1A and KDM1B can remove mono and dimethyl groups but not trimethyl groups [27][3]. The Jumonji-C domain-containing histone demethylases, the second group of KDMs, can remove trimethyl groups from histone lysine residues, as well as di and monomethyl groups [27][3]. These two KDM categories are further subdivided into KDM2, KDM3, KDM4, KDM5 and KDM6, with each demethylating a specific lysine residue [27][3]. In embryonic stem cells, KDM1A (also known as lysine specific demethylase 1 (LSD1)) expression levels are typically elevated, but are reduced during differentiation [32][8]. LSD1 was also reported to maintain the balance between H3K4 di/tri methylation and H3K27 trimethylation marks (bivalent heterochromatin) at regulatory regions of several developmental genes, thus suppressing differentiation-associated genes and maintaining pluripotency in embryonic stem cells [33][9]. LSD1 has been shown to be an important regulator in maintaining the CSC population in treatment-resistant breast cancer cell lines. Pretreatment with LSD1 inhibitors improved treatment sensitivity to doxorubicin treatment [34][10]. Increased levels of KDM5A and KDM5B have been associated with chemoresistance in cancer and appear to contribute to an increase in cancer cell proliferation [35][11]. High levels of KDM5B are associated with the repression of tumor suppressor genes and apoptosis-related genes [36][12]. DICER, an enzyme involved in processing of microRNAs that are regulating the EMT pathway, showed a decrease in expression in breast cancer cell lines after hypoxic exposure, even though KDM6A and KDM6B were enriched in the promoter region, highlighting the importance of oxygen to carry out demethylating activity of KDM6A and KDM6B [37][13]. KDM6A was inactivated by a hypoxic environment in myoblast cell line C2C12, resulting in sustained H3K27me3, preventing myogenic differentiation [38][14]. Histone deacetylases (HDACs) are another group of eraser enzymes that remove acetyl groups from histones, which leads to chromatin compaction. As a result, DNA becomes inaccessible to transcription factors for gene expression. These enzymes cannot bind to DNA directly and require repressor complexes to facilitate DNA binding, such as NuRD (nucleosome remodeling and deacetylase complex), CoREST (co-repressor for element-1-silencing transcription factor) and KDM [27][3]. Knockdown experiments reveal that HDAC7 epigenetically modifies transcription start sites and super-enhancers of oncogenes such as C-MYC, CD44 and BMI-1 and reduces the expression of CD49f in breast cancer stem cells [39][15]. A knockdown of HDAC7 led to a reduction of sphere-forming ability and in vivo tumor growth of classic CSC phenotypes, and HDAC7 levels of expression in CSCs were also found to be higher than in non-stem cancer cells [40][16]. Being downstream of class 1 and class 2 HDACs, HDAC7 could be important in a targeting strategy for eliminating breast and ovarian CSCs [39][15]. Class 3 HDACs, also known as sirtuins, are dependent on NAD+ (nicotinamide adenine dinucleotide) for their activity thus playing a role in the metabolic regulation of the cell [27][3]. Class 4 HDACs include HDAC11, which has also been correlated with increased expression in lung cancer stem cell lines and regulates the expression of SOX2 [(sex determining region Y)-box 2], an important transcription factor to maintain CSC self-renewal [41][17]. DNA demethylation proteins are a third type of eraser involved in the oxidation of 5-methylcytosine (5mC), present in CG dinucleotides, into 5 hydroxymethylcytosine [27][3]. TET (Ten Eleven Translocation) demethylating proteins fall under this group of epigenetic regulators. TET1, TET2 and TET3 play pivotal roles in the development and maintenance of the stem cell phenotype [27][3]. TET2 was found to be involved in the regulation of genes associated with self-renewal and differentiation in hematopoietic stem cells [42][18], and mutations in TET2 have been associated with hematological malignancies [43][19].

2.3. Reader Enzymes

Readers are proteins that recognize histone and DNA modifications and can sense chromatin conformation. Bromodomain belongs to bromodomain and extra-terminal domain (BET) family members and recognizes acetylated lysine [44][20]. BRD4 (bromodomain-containing protein 4), which belongs to the BET family, binds to an acetylated promoter, allowing transcription of target genes [44][20]. C-MYC expression is regulated by BRD4 binding to the promoter and enhancer [22][21]. C-MYC was targeted using a BRD4 inhibitor, JQ1, in medulloblastoma. Inhibition of BRD4 was found to downregulate transcription of C-MYC, which led to a further reduction in cell growth as well as cell cycle arrest at the G1 phase. Stem cell-associated pathways were found to be downregulated upon treatment with JQ1. NANOG, Nestin and SOX2 classical stem cell markers were downregulated, while MAP2 (microtubule associated protein 2), a differentiation marker for neurons, was found to be upregulated [45][22]. C-MYC was validated as the target of BRD4 using luciferase-expressing plasmids controlled by C-MYC, studied in Daoy cells. ThIt is work  showed a decrease in luciferase activity in the presence of JQ1 [45][22]. In another study, BRD4 was found to be associated with the N-termini of TWIST and WNT5A gene expression which are essential regulators of the EMT pathway [46][23].

3. Other Regulators of Cancer Stem Cells

3.1. Long Non-Coding RNA

A large part of the human genome contains DNA that does not code for any proteins and was long assumed to be “junk DNA”. Over the years, research has uncovered that although a large portion of transcribed RNA does not code for proteins, it serves a vital function in the regulation of genes [47][24]. Many of these regulatory segments fall into the category of long non-coding RNAs (lncRNA), which are greater than 200 nucleotides in length. LncRNA can be found in intergenic regions, transcribed from introns, sense RNA or antisense RNA [48][25]. LncRNAs can act as decoys, guides and signaling molecules. Decoys can cause gene repression by blocking the binding of proteins to RNA, inhibiting transcription [47,49][24][26]. Alternately, guides can facilitate gene expression by helping transcription factors or multiprotein complexes such as PRC (polycomb repression complex) to bind to target genes [47][24]. LncRNAs can also play integral roles in the regulation of CSCs. In the case of liver cancer stem cells, increased expression of lncTCF7 led to the activation of the Wnt pathway through the recruitment of SWI/SNF to the promoter of TCF [50][27]. In embryonic stem cells, lncRNAs were found to be important targets of the transcription factors OCT4, SOX2, C-MYC, KLF4 (Krueppel-like factor 4) and NANOG, which are involved in maintaining pluripotency [51][28]. Knocking down lncTCF7 expression caused a reduction in the expression of OCT4, SOX2, C-MYC, KLF4 and NANOG, along with reduced sphere-forming ability of liver cancer stem cells [50][27]. High XIST (LncRNA X inactive specific transcript) expression, a long non-coding RNA, was correlated with low miR-200c expression levels, and was identified as a potential target to eradicate bladder cancer stem cells [52][29].

3.2. ATP-Dependent Chromatin-Remodeling Complexes

Dynamic modification of chromatin is performed by ATP-dependent chromatin-remodeling complexes that utilize ATP to slide (translocate or move the histone along the DNA), evict or replace nucleosomes, thereby affecting gene expression [53][30]. Depending on the catalytic unit and associated subunits, the four classes of chromatin remodelers are SWI/SNF, CHD, ISWI and INO80. These four classes of remodelers possess an ATPase domain which is conserved across eukaryotes and function as multi-subunit complexes in association with tissue-specific subunits [54,55][31][32]. Epigenetic modification occurs through the binding of these complexes to specific chromatin domains such as bromo, chromo and SANT domains [56][33].

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