Histone modifications as a pathogenic mechanism of colorectal tumorigenesis

The International Journal of Biochemistry & Cell Biology 44 (2012) 1276–1289

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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel

Review

Histone modifications as a pathogenic mechanism of colorectal tumorigenesis Antonios N. Gargalionis 1 , Christina Piperi 1 , Christos Adamopoulos, Athanasios G. Papavassiliou ∗ Department of Biological Chemistry, University of Athens, Medical School, 11527 Athens, Greece

a r t i c l e

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Article history: Received 30 March 2012 Received in revised form 2 May 2012 Accepted 2 May 2012 Available online 11 May 2012 Keywords: Colorectal cancer Epigenetics Histone acetylation Methylation Tumorigenesis

a b s t r a c t Epigenetic regulation of gene expression has provided colorectal cancer (CRC) pathogenesis with an additional trait during the past decade. In particular, histone post-translational modifications set up a major component of this process dictating chromatin status and recruiting non-histone proteins in complexes formed to “handle DNA”. In CRC, histone marks of aberrant acetylation and methylation levels on specific residues have been revealed, along with a plethora of deregulated enzymes that catalyze these reactions. Mutations, deletions or altered expression patterns transform the function of several histonemodifying proteins, further supporting the crucial role of epigenetic effectors in CRC oncogenesis, being closely associated to inactivation of tumor suppressor genes. Elucidation of the biochemical basis of these new tumorigenic mechanisms allows novel potential prognostic factors to come into play. Moreover, the detection of these changes even in early stages of the multistep CRC process, along with the reversible nature of these mechanisms and the technical capability to detect such alterations in cancer cells, places this group of covalent modifications as a further potential asset for clinical diagnosis or treatment of CRC. This review underlines the biochemistry of histone modifications and the potential regulatory role of histone-modifying proteins in CRC pathogenesis, to date. Furthermore, the underlying mechanisms of the emerging epigenetic interplay along with the chemical compounds that are candidates for clinical use are discussed, offering new insights for further investigation of key histone enzymes and new therapeutic targets. © 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1277 Biochemical basis of post-translational histone modifications in CRC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1277 Mechanisms of acetylation marks in CRC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278

Abbreviations: AP2A1, adaptor related protein complex 2, subunit alpha 1; APAF1, apoptotic protease activating factor 1; APC, adenomatous polyposis coli; BRCA1, 2, breast cancer 1, 2; CARM1, co-activator-associated arginine methyltransferase 1; CBP, cAMP-responsive element binding (CREB)-binding protein; CDO1, cysteine dioxygenase, type I gene; CDC2, cell division cycle-2; C/EBP, CCAAT enhancer-binding protein; ceRNAs, competitive endogenous RNAs; DMAP1, DNA methyltransferase 1-associated protein 1; DNMTs, DNA methyltransferases; DNMT1, 3B, DNA methyltransferase 1, 3B; ECEL1, endothelin-converting enzyme-like 1; EED, embryonic ectoderm development protein; EMT, epithelial to mesenchymal transition; EZH2, enhancer of zeste homolog 2; 5FU, 5-fluorouracil; G9a, lysine 9 (K9) of histone H3-specific methyltransferase; HATs, histone acetyltransferases; HDACs, histone deacetylases; HDMs, histone demethylases; HMTs, histone methyltransferases; HOTAIR, Hox transcript antisense intergenic RNA; HSPC105, short-chain dehydrogenase/reductase family 42E mem; JHDMs, Jumonji C-domain containing histone demethylases; JMJD1A, Jumonji-domain containing 1A; K, lysine; KLF4, Krüppel-like factor 4; 15-LOX-1, 15-lipoxygenase-1; LSD1, lysine-specific demethylase 1; MAGEA3, melanoma-associated antigen 3; MBD1, 2, 3, methylCpG-binding domain protein 1, 2, 3; MeCP2, methyl-CpG-binding protein 2; miRNAs, microRNAs; MLH1, MutL homolog 1; MLL1, 3, myeloid/lymphoid leukemia 1, 3; MREs, miRNA response elements; MSI, microsatellite instability; ncRNAs, non-coding RNAs; NDRG1, N-myc downstream-regulated gene 1; NEURL, neuralized-like protein 1; NF-␬B, nuclear factor kappa B; PCAF, p300/CBP-associated factor; PIK3CB, phosphoinositide-3-kinase, catalytic, beta polypeptide; PPAR␥, peroxisome proliferator-activated receptor gamma; PRC1, 2, Polycomb repressive complex 1, 2; PRDM, PR domain zinc finger proteins; PRMT1, 5, protein arginine methyltransferase 1, 5; PSMD9, 26S proteasome non-ATPase regulatory subunit 9; PTEN, phosphatase and tensin homolog; R, arginine; RGC32, response gene to complement 32; RIZ1, retinoblastoma protein-interacting zinc finger; RUNX3, runt-related transcription factor 3; SAHA, suberoylanilide hydroxamic acid; siRNA, small interfering RNA; SIRT1, sirtuin 1; SMYD3, SET and MYND domain-containing protein 3; STAT1, 3, signal transducer and activator of transcription 1, 3; SUV39H1, histone-lysine N-methyltransferase; SUZ12, suppressor of zeste 12 homolog; TCF12, transcription factor 12; TIMP3, tissue inhibitor of metalloproteinase 3; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TSA, trichostatin A; VEGFR1, vascular endothelial growth factor receptor 1; Wnt10B, wingless-type MMTV integration site family, member 10B; XAF1, X-linked inhibitor of apoptosis-associated factor 1; ZEB1, zinc finger E-box-binding homeobox 1. ∗ Corresponding author at: Department of Biological Chemistry, Medical School, University of Athens, 75, M. Asias Street, 11527 Athens, Greece. Tel.: +30 210 746 2508/9; fax: +30 210 779 1207. E-mail addresses: [email protected], [email protected] (A.G. Papavassiliou). 1 Equal contribution. 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2012.05.002

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5. 6.

7. 8. 9.

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Regulatory role of histone acetylation proteins in CRC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279 4.1. HATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279 4.1.1. The CBP/p300 family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279 4.1.2. The GNAT and MYST families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279 4.2. HDACs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1280 4.2.1. Class I of HDACs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1280 4.2.2. Classes II and III of HDACs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1281 Mechanisms of methylation marks in CRC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282 Regulatory role of histone methylation proteins in CRC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282 6.1. Lysine HMTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282 6.2. Arginine HMTs and HDMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1283 Epigenetic alterations interplay in CRC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284 Regulation of histone modifications in clinical practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284 Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286

1. Introduction Being one of the most common types of malignancy, along with one of the most frequent etiologies of cancer mortality in both genders worldwide, colorectal cancer (CRC) still presents a major public health problem to confront and a rapidly evolving research field to keep up with (Jemal et al., 2011; Siegel et al., 2012). Nevertheless, it is well established that sporadic and hereditary CRC constitute the histological and clinical outcome of a multistage, though hierarchically structured, genetic process characterized by the sequential accumulation of genetic and epigenetic alterations (Fearon and Vogelstein, 1990). CRC genetic alterations present a well studied field over the past three decades providing an overall paradigm of the multistep carcinogenesis process. Driver mutations affect major intracellular signaling pathways implicated in proliferation, differentiation, cell adhesion and migration, apoptosis, DNA stability and repair (Saif and Chu, 2010). Focusing on epigenetic alterations, a recently evolving research area, accumulating data indicate an additional trait of CRC pathogenesis. The term epigenetics refers to heritable changes that although not affecting DNA sequence, they play a critical regulatory role in gene expression. Epigenetic changes include DNA methylation, loss of imprinting, post-translational histone modifications, nucleosome positioning, chromatin looping and small non-coding RNAs interference (van Engeland et al., 2011). Among these alterations, the most extensively characterized is aberrant DNA methylation including both global DNA hypomethylation, an age-dependent process with poor prognosis occurring at early stages in CRC (Suzuki et al., 2006), and CpG island hypermethylation, presenting an additional hit in the classic Knudson genetic model (Knudson, 2001) for inactivation of tumor suppressor genes (Herman and Baylin, 2003), with main example the mismatch repair gene MutL homolog 1 (MLH1) implicated in CRC pathogenesis (Herman et al., 1998). Although individually studied, a complex interplay has emerged between DNA methylation and histone modifications that is mediated by biochemical interactions of histone and DNA methyltransferases (HMTs and DNMTs, respectively) with the recruitment of histone deacetylases (HDACs) (Cedar and Bergman, 2009; Tachibana et al., 2008; Zhao et al., 2009). Taking all these into account, an additional epigenetic phenotype has been attributed to CRC that is critically regulated by the post-translational modifications of histone residues, allowing the generation of a corresponding multistep epigenetic CRC model, with potential novel therapeutic targets. The present review explores the biochemistry behind histone modifications and the respective regulatory role of histonemodifying proteins in CRC pathobiology. In addition, the underpinning mechanisms of the emerging epigenetic interplay along

with targeted therapy in research are discussed, providing new insights for further investigation of pivotal histone enzymes and new mechanisms in favor of pharmaceutical treatment. Although the types of histone modifications will be described in separate sections for comprehensive purposes, one should bear in mind that these changes and the underlying mechanisms take place simultaneously or in parallel and are exposed to a constant and mutual regulation. 2. Biochemical basis of post-translational histone modifications in CRC The basic nucleosome unit is composed of four core histone proteins, H2A, H2B, H3 and H4, that form an octamer around which a segment of DNA winds with 147 base pairs in 1.67 left-handed superhelical turns. Highly basic histone N-terminal domains are able to protrude from the nucleosome establishing contact with adjacent ones (Fig. 1). At least eight different types of modifications have been characterized on multiple sites of specific residues of the “free” N-terminal domains, notably lysine (K) and arginine (R). These include acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deimination and proline isomerization. Acetylation and methylation constitute the vast majority of known modifications, being mediated by a number of specialized enzymes. Their functional role relies on the disruption of chromatin contacts and recruitment of non-histone proteins, thus defining chromatin’s proper structure and allowing protein complex formation for DNA “handling” (Cosgrove et al., 2004; Kouzarides, 2007). The emerging variability on the type of modified residue, the type and number of modifications, or the different modifying proteins as well as the immediate interplay of these changes, gives us a hint of the underlying complexity that this mechanism involves. In CRC, aberrant histone modification patterns have been detected, being generated by a plethora of deregulated enzymes. Mutations, deletions or altered expression profiles alter the function of several histone-modifying proteins, thus supporting the major role of epigenetic effectors in CRC tumorigenesis (Ellis et al., 2009). These events contribute to cancer initiation and progression by altering the physiological levels of gene expression–mostly by inactivating tumor suppressor genes (Konishi and Issa, 2007)–and by inducing genome instability due to direct effects in the higher order of chromatin, chromosome condensation and mitotic disjunction (Bannister and Kouzarides, 2011). Consequently, a new terminology, “histo-oncomodifications”, has evolved describing the histone covalent alterations that have been linked to cancer (Fullgrabe et al., 2011). The participation of these changes even in early stages of oncogenesis, their reversible nature and the technical ability to detect such alterations in neoplasmatic cells places this

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Fig. 1. Aberrant histone modification marks implicated in colorectal cancer (CRC) pathogenesis. The core nucleosomal structure with gain (cross) or loss (rectangular) of main acetylation (Ac) and methylation (Me) marks on specific histone residues involved in CRC process is shown. Up-regulating (green) or down-regulating (red) properties of these marks over histone-modifying proteins (oval shapes) and tumor-associated or suppressor gene expression (top end) are indicated along with the global effect of RGC32 gene deregulation over multiple histone acetylation (bottom end). CARM1, co-activator-associated arginine methyltransferase 1; CDO1, cysteine dioxygenase, type I gene; EZH2, enhancer of zeste homolog 2; G9a, lysine 9 (K9) of histone H3-specific methyltransferase; H1-4, histones 1-4; HSPC105, short-chain dehydrogenase/reductase family 42E mem; K, lysine; 15-LOX-1, 15-lipoxygenase-1; LSD1, lysine-specific demethylase 1; MAGEA3, melanoma-associated antigen 3; MLL3, myeloid/lymphoid leukemia 3; NH2 , amino terminus; NDRG1, N-myc downstream-regulated gene 1; PIK3CB, phosphoinositide-3-kinase, catalytic, beta polypeptide; PPAR␥, peroxisome proliferatoractivated receptor gamma; R, arginine; RGC32, response gene to complement 32; RIZ1, retinoblastoma protein-interacting zinc finger; SIRT1, sirtuin 1; SMYD3, SET and MYND domain-containing protein 3; SUV39H1, histone-lysine N-methyltransferase; Wnt10B, wingless-type MMTV integration site family, member 10B.

group of chemical changes as a further potential asset for clinical diagnosis or treatment of CRC.

3. Mechanisms of acetylation marks in CRC Acetylation takes place in lysine residues of the four core histones, balanced by the opposite effects of two main enzymatic groups, the histone acetyltransferases (HATs) and the HDACs. H3 and H4 histone acetylation levels are crucial regarding chromatin status and the regulation of gene expression, being generally associated with active gene transcription (Kouzarides, 2007). Functionally, an adequate acetylation levels either change the electrostatic charge of histones and therefore the chromatin structure (cis mechanism), or operate as binding sites for bromodomain protein recognition motifs specialized to recognize acetylated lysine residues (trans mechanism) (Fullgrabe et al., 2011; Wang et al., 2007). Implication of these conserved domains in binding of acetylated, non-histone polypeptides (such as transcription factors) as well as histones highlights the role of acetylation in regulating intracellular signaling. Therefore, many HATs have been redefined as co-factor acetyltransferases due to their targeting several transcription factors (e.g. p53, erythroid transcription factor [GATA-1], Krüppel-like factor) (Bannister and Miska, 2000). Aberrant acetylation marks have been associated with CRC pathogenesis (Fig. 1; Table 1). Among the first histone marks that

were reported to be deregulated in CRC was the global loss of acetylation of histone H4 at lysine 16 (H4K16ac), an established common hallmark in various types of malignancy (Fraga et al., 2005). A reduction in H4K16 acetylation has been observed in CRC cell lines as well as in primary tumors. Acetylation levels were found reduced from 60% in normal mucosa to 35% in the primary tumor, potentially leading to a more “loose” chromatin structure that contributes to genome instability. Likewise, global hypoacetylation of H4K12 and H3K18 correlated with poorly differentiated colorectal adenocarcinomas (Ashktorab et al., 2009). By contrast, net or global acetylation of H3 and H4 was augmented in well and moderately differentiated tumors, progressively increasing from normal tissue to carcinoma as evidenced for the H4K12 mark. An additional residue modification, H3K9 hypoacetylation, correlated with the histological type in metachronous CRC liver metastasis, whereas deacetylation of the same residue was further linked to inactivation of the tumor suppressor gene E-cadherin in CRC cell lines (Liu et al., 2008; Tamagawa et al., 2012). Interestingly, in a CRC cell line stably transformed with the oncogenic form (mutated) of Harvey-Ras protein that triggers epithelial to mesenchymal transition (EMT), global H3K9/14 acetylation was observed in the promoters of E-cadherin and Cyclin D1 (an EMT and a cell-cycle gene, respectively) that induced down-regulation of the corresponding protein expression via the Ras signaling pathway (Pelaez et al., 2010). Elevation of global H3 acetylation was accompanied by gene reactivation and localized DNA hypomethylation

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Table 1 Histone acetylation marks in CRC. Acetylation marks

Impaired function

Gene regulation

References

H2AK5ac, H2BK5ac, H2BK15ac Global H3ac

Hypoacetylation (CRC cell lines) Hypoacetylation (CRC primary tumors) Hyperacetylation (CRC cell lines) Hypoacetylation (CRC liver metastasis, CRC cell lines, Harvey-Ras CRC cell lines)

RGC32 induced p21WAF1 down-regulation 15-LOX-1 reactivation E-cadherin and Cyclin D1 promoters, RGC32 induced

Hypoacetylation (CRC primary tumors, CRC cell lines) Hypoacetylation (CRC metastatic cell lines) Hyperacetylation (CRC cell lines) Hypoacetylation (CRC cell lines) Hyperacetylation (CRC primary tumors) Hypoacetylation (grade III CRC primary tumors) Hypoacetylation (CRC primary tumors, CRC cell lines)

RGC32 induced

Vlaicu et al. (2010) Chen et al. (2007) Zuo et al. (2008) Liu et al. (2008), Pelaez et al. (2010), Tamagawa et al. (2012), Vlaicu et al. (2010) Ashktorab et al. (2009), Vlaicu et al. (2010) Li and Chen (2011) Zuo et al. (2008) Vlaicu et al. (2010) Ashktorab et al. (2009)

H3K9/14ac

H3K18ac Global H4ac H4K8 H4K12ac

H4K16ac

after treatment with 5-aza-2 -deoxycytidine, an inhibitor of DNMT (Mossman and Scott, 2011). Additionally, deregulation of the response gene to complement 32 (RGC32), a cell-cycle activator and cell division cycle-2 (CDC2) substrate found in various human malignancies, seems to have as target the aberrant acetylation of multiple histone proteins contributing to CRC (Vlaicu et al., 2010). Furthermore, H3 and H4 acetylation is crucial for transcriptional activation of 15-lipoxygenase-1 (15-LOX-1), whose product is silenced in CRC cells (Zuo et al., 2008). Down-regulation of p21WAF1 transcription that is also involved in CRC has been associated with H3 hypoacetylation and histone enzymes (Chen et al., 2007). Finally, considering CRC metastasis, reduced H4 acetylation was associated with the silencing of N-myc downstream-regulated gene 1 (NDRG1), a specific metastasis suppressor gene, in the highly metastatic cell line SW620, when compared to SW480 cells that possessed higher levels of H4 acetylation (Li and Chen, 2011). 4. Regulatory role of histone acetylation proteins in CRC 4.1. HATs HATs are largely known as important transcription co-activators recruited by transcription factors, capable to acetylate histones and non-histone proteins (Grunstein, 1997; Santos-Rosa and Caldas, 2005; Yang, 2004) (Fig. 2). HATs recruit acetyl-CoA in order to catalyze an acetyl group’s transfer to lysine side chain’s ␧-amino group, thus converting the lysine’s positive charge into neutral and, consequently, reducing histone–DNA affinity. They are divided into three main families: the cAMP-responsive element binding (CREB)-binding protein (CBP)/p300 family, the GNAT family (p300/CBP-associated factor [PCAF]/GCN5 N-acetyltransferase) and the MYST family (MOZ, YBF2, SAS2, Tip60), mostly found inactivated in cancer by gene mutations or being deregulated by viral oncoproteins (Wang et al., 2007) (Table 2). 4.1.1. The CBP/p300 family The CBP/p300 family members regulate gene transcription through chromatin remodeling and are implicated in cell proliferation and differentiation processes (Iyer and Ozdag, 2004). Missense mutations generating a truncated p300 protein have been identified in primary CRC tumors and CRC cell lines, attributing a tumor suppressor role to p300 gene (Davis and Brackmann, 2003; Gayther et al., 2000; Miremadi et al., 2007). This is further confirmed by mutations (mononucleotide repeats) detected in both p300 and CBP genes of CRC cell lines with microsatellite instability (MSI) as well as in a small number of primary tumors with MSI (Ionov et al., 2004).

NDRG1 suppression 15-LOX-1 reactivation RGC32 induced

Fraga et al. (2005)

Additionally, p300/CBP/PCAF inactivating truncating mutations and CBP intronic microdeletion have been identified in several CRC cell lines. However, no somatic mutations were found in any primary CRC tumors, although there was a p300 loss of heterozygosity in 38% of them (Bryan et al., 2002; Ozdag et al., 2002). This loss of heterozygosity of p300 was further coupled to a missense mutation found in colorectal carcinoma (Muraoka et al., 1996). Regarding germline mutations, none were detected in p300 of breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) negative families with breast cancer and family history of CRC tumors (Campbell et al., 2004). Functional properties of p300 and CBP involve interaction with and acetylation of the transcription factor Krüppel-like factor 4 (KLF4) that is implicated in both proliferation and differentiation in CRC. In vitro and in vivo studies have shown a dual role for KLF4 in activating or repressing transcription, depending on its interaction with co-activators (CBP/p300) or co-repressors (HDAC3), respectively (Evans et al., 2007). The same proteins function as potential co-factors regulating the Snail, zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin and vitamin D receptor interactions in CRC, thus being critical in EMT and tumor progression processes (Pena et al., 2006). CBP interacts also physically with the nuclear protein X-linked inhibitor of apoptosis-associated factor 1 (XAF1) potentiating cell growth inhibition through adaptor related protein complex 2, subunit alpha 1 (AP2A1) (Sun et al., 2008). Patterning of CBP/p300 expression in CRC tissues needs further investigation since contradictory results are available. Although low expression levels of p300 were found in tumor tissue compared to normal mucosa by Giannini and Cavallini (2005), a different study indicated overexpression of both proteins being correlated with poor prognosis and, surprisingly, high expression levels of CBP being associated with long-term survival (Ishihama et al., 2007). 4.1.2. The GNAT and MYST families The GNAT family of HATs comprises the enzymes PCAF and GCN5, which exhibit increased activity on H3 that has been previously phosphorylated in Ser 10. A recent in vitro study further implicates activation of the B-Raf oncogene in the regulation of these enzymes (Pelaez et al., 2010). Moreover, mRNA levels of PCAF as co-activator of estrogen receptor gene expression were found significantly down-regulated in cancerous tissue compared to adjacent normal colorectal mucosa, indicating its implication in the development of CRC (Giannini and Cavallini, 2005). PCAF has also been associated with the regulation of ˇ-catenin gene expression. Knockdown of PCAF in colorectal cells was correlated with reduced transcriptional activity, protein and acetylation levels of

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Fig. 2. Suggested mechanisms of deregulated histone modifications and their modifying proteins in colorectal cancer (CRC) progression. Under physiological conditions, a euchromatin state (upper part) is established that allows HATs (green) to interact with TFs (yellow) in order to regulate gene expression homeostasis. Active acetylation marks (light green croos) induced by HATs along with balanced active (green cross) and inactive methylation marks (dark red rectangular) governed by HMTs (blue) co-regulate the process. During CRC progression, deregulated histone modification patterns generate a compact heterochromatin state (lower part). HDACs (red) are recruited to form inactive hypoacetylation marks (red rectangular), whereas aberrant HMTs (blue) and their corresponding methylation marks (green/dark red crosses) promote repression of tumor suppressor genes and oncogenic activity, respectively. These processes are directly linked to the hallmarks of CRC aggressive phenotype. Ac, acetylation; CAs, co-activators; CRs, co-repressors; DNMTs, DNA methyltransferases; HATs, histone acetyltransferases; HDACs, histone deacetylases; HMTs, histone methyltransferases; Me, methylation; MSI, microsatellite instability; PPAR␥, peroxisome proliferator-activated receptor gamma; TFs, transcription factors.

␤-catenin, further promoting cell differentiation, inhibition of cell migration and repression of tumor growth in nude mice (Ge et al., 2009). Moving to the MYST family members, a main representative, the Tip60 HAT, is responsible for cellular response to DNA damage, functioning as co-activator for key proteins such as nuclear factor kappa B (NF-␬B) (Avvakumov and Cote, 2007), nuclear hormone receptors (Brady et al., 1999; Gaughan et al., 2001, 2002), ␤-catenin (Sierra et al., 2006), c-Myc oncoprotein (Frank et al., 2003) and E2F (Taubert et al., 2004). In CRC tissues, Tip60 was significantly down-regulated (Gorrini et al., 2007; Lleonart et al., 2006) suggesting a tumor suppressing role. Furthermore, immunohistochemical delocalization of Tip60 to the cytoplasm along with its down-regulation were statistically significantly correlated with basic parameters of the CRC aggressive phenotype (Sakuraba et al., 2009). Moreover, a defect in the ratio of Tip60/p400 expression levels (with p400 being an ATPase present in a complex with Tip60 responsible for nucleosomal incorporation of histone variants) has been found critical for CRC cell proliferation through DNA damage response pathways, particularly in response to first line 5-fluorouracil (5FU) treatment. When the ratio is reversed with Tip60 being overexpressed, increased apoptosis and decreased proliferation were detected (Mattera et al., 2009). Tip60 may also participate indirectly in various mechanisms of CRC tumorigenesis due to its interaction with crucial “oncogenic” factors, such as NF-␬B and p53 (Avvakumov and Cote, 2007; Lleonart et al., 2006).

4.2. HDACs HDACs, in turn, have aroused great interest as therapeutic target molecules since the removal of acetyl groups in histone lysine residues and non-histone substrates enhances DNA condensation and represses tumor suppressor gene transcription (Bolden et al., 2006). Several HDACs have been found up-regulated in CRC, while their down-regulation inhibits tumor growth establishing their corepressing role on tumor suppressor genes (Mariadason, 2008a; Fig. 2; Table 2). HDACs are divided into four classes: class I constituting HDACs 1, 2, 3, 8 that are localized to the nucleus, class II comprised of HDACs 4, 5, 6, 7, 9, 10 present both in the nucleus and the cytoplasm, class III or sirtuins 1–7 and class IV composed of HDAC 11 (Federico and Bagella, 2011).

4.2.1. Class I of HDACs Class I HDACs are commonly overexpressed in the proliferative compartment of normal colon as well as in CRC human tumors (Giannini and Cavallini, 2005; Huang et al., 2005; Ishihama et al., 2007; Nakagawa et al., 2007). HDAC1 up-regulation has been significantly correlated with the depth of tumor invasion, the stage of the disease and the five-year survival rates constituting a potential prognostic marker along with HDACs 2 and 3, which revealed a similar pattern of behavior (Ashktorab et al., 2009; Higashijima et al., 2011; Weichert et al., 2008). However, a single study employing quantitative real-time polymerase chain reaction indicates low

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Table 2 HATs and HDACs impaired functions in CRC.

HATs p300

CBP

Impaired function

References

Truncating mutations (CRC primary tumors, CRC cell lines) Truncating mutations (MSI CRC primary tumors, MSI CRC cell lines) Loss of heterozygosity (CRC primary tumors) Underexpression (CRC primary tumors) Overexpression (CRC primary tumors)

Bryan et al. (2002), Davis and Brackmann (2003), Gayther et al. (2000), Miremadi et al. (2007), Muraoka et al. (1996) Ionov et al. (2004) Bryan et al. (2002), Muraoka et al. (1996) Giannini and Cavallini (2005) Ishihama et al. (2007)

Truncating mutations (CRC cell lines) Truncating mutations (MSI CRC primary tumors, MSI CRC cell lines) Intronic microdeletion-exonic deletion (CRC cell lines) Overexpression (CRC primary tumors)

Ozdag et al. (2002) Ionov et al. (2004) Ozdag et al. (2002) Ishihama et al. (2007)

GCN

B-Raf mediated up-regulation (CRC cell lines)

Pelaez et al. (2010)

PCAF

Truncating mutations (CRC cell lines) B-Raf mediated up-regulation (CRC cell lines) Underexpression (CRC primary tumors)

Ozdag et al. (2002) Pelaez et al. (2010) Giannini and Cavallini (2005)

Tip60

Underexpression (CRC primary tumors) Cytoplasm delocalization (CRC primary tumors)

Gorrini et al. (2007), Lleonart et al. (2006), Sakuraba et al. (2009)

Overexpression (CRC primary tumors)

Giannini and Cavallini (2005), Higashijima et al. (2011), Huang et al. (2005), Ishihama et al. (2007), Nakagawa et al. (2007), Weichert et al. (2008) Ozdag et al. (2006)

HDACs Class I HDAC1

Underexpression (CRC primary tumors) HDAC2

Truncating mutation (CRC primary tumors, CRC cell line) Overexpression (CRC primary tumors, CRC APC-deficient mice)

Ozdag et al. (2006), Ropero et al. (2006) Ashktorab et al. (2009) Giannini and Cavallini (2005), Huang et al. (2005), Nakagawa et al. (2007), Weichert et al. (2008), Zhu et al. (2004)

HDAC3

Overexpression (CRC primary tumors, mouse intestinal adenomas)

HDAC8

Overexpression (CRC primary tumors)

Giannini and Cavallini (2005), Nakagawa et al. (2007), Weichert et al. (2008), Wilson et al. (2006) Nakagawa et al. (2007)

Class II HDAC4 HDAC5 HDAC7A

Down-regulation (CRC primary tumors) Underexpression (CRC primary tumors) Overexpression (CRC primary tumors)

Lleonart et al. (2006) Ozdag et al. (2006) Ozdag et al. (2006)

Underexpression (CRC high grade, CRC primary tumors) Overexpression (CRC cell line, MSI + CpG island methylation CRC primary tumors)

Kabra et al. (2009) Ozdag et al. (2006) Kabra et al. (2009), Kuzmichev et al. (2005), Nosho et al. (2009)

Class III SIRT1

mRNA levels of HDAC1 in CRC tumors compared to normal colorectal tissue (Ozdag et al., 2006). A robust expression of HDAC3 has also been observed in mouse intestinal adenomas, confined to cryptic epithelium, which is the proliferative compartment of the tissue (Wilson et al., 2006). Mechanistic studies have shown that silencing of HDACs 1, 2 and 3 genes results in growth and cell survival inhibition, whereas their overexpression hampers basal and butyrate-induced p21 transcription in a Sp1/Sp3-dependent manner (Spurling et al., 2008; Wilson et al., 2006). More specifically, deregulation of HDAC2 that leads to elevated expression has been previously reported in CRC tumorigenesis, being induced by the loss of the adenomatous polyposis coli (APC) tumor suppressor gene, depending on Wnt and c-Myc signaling pathways (Zhu et al., 2004). The oncogenic role of HDAC2, partially through p21 repression (Huang et al., 2005), was confirmed in vivo, when LacZ-HDAC2 knockout mice crossed with tumor-proned APCmin mice formed shortened crypts and villi in addition to lower tumor rates (Zimmermann et al., 2007). Moreover, a single nucleotide deletion was detected in HDAC2 screening of CRC cell lines causing a frameshift starting at amino acid 543 and leading to the addition of 16 amino acids to the C-terminus of the protein (Ozdag et al., 2006). HDAC2 mutation participates to the apoptotic resistance mechanisms of CRC cells to HDAC inhibitors mediated by the apoptotic protease activating factor 1 (APAF1) (Hanigan et al., 2008; Mariadason, 2008b). The presence of an inactivating mutation was also demonstrated for HDAC2 in CRC cell

lines with MSI and primary tumors, conferring resistance to trichostatin A (TSA), a known HDAC inhibitor (Ropero et al., 2006). Finally, long-term knockdown of HDAC3 has been found to influence Wnt pathways by suppressing ␤-catenin’s translocation to the nucleus and increasing expression of Wnt inhibitors (Godman et al., 2008). 4.2.2. Classes II and III of HDACs Class II and III HDACs are commonly down-regulated in CRC compared to normal colon as indicated by most studies (Lleonart et al., 2006; Wilson et al., 2008). HDAC4 is expressed in the proliferative zone and intervenes with cell differentiation by repressing p21 gene expression (Wilson et al., 2008). In accordance, mRNA levels of HDAC5 were reduced in colon carcinoma relative to normal tissue whereas the same study indicates an overexpression of HDAC7A mRNA in CRC (Ozdag et al., 2006). Among the sirtuin (class III) family members, sirtuin 1 (SIRT1) presents the most extensively studied therapeutic target being involved in control of cell differentiation. Its expression is primarily elevated in cancer cells (Kuzmichev et al., 2005) with only one study showing reduced mRNA levels in CRC tumors compared to normal tissue (Ozdag et al., 2006). However, overexpression of SIRT1 was correlated with high MSI and CpG island methylation phenotype in CRC (Nosho et al., 2009). SIRT1 knockdown accelerated xenograft oncogenesis in HCT116 cells, whereas its overexpression inhibited the process. It is important to note that SIRT1’s inhibition sensitized CRC cells to apoptosis by chemotherapeutic drugs.

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Immunohistochemical analysis revealed increased SIRT1 levels in normal colon and benign adenomas being relatively absent from high grade tumors, indicating an ambiguous role for SIRT1 with pleiotropic effects during carcinogenesis (Kabra et al., 2009). Another study points toward SIRT1’s ability to induce upregulation of H3K9 and H3K14 acetylation at silenced tumor suppressor gene promoters followed by gene reactivation (Pruitt et al., 2006). In addition, RGC32 knockdown was associated with SIRT1 down-regulation. A positive correlation was demonstrated between the nuclear presence of SIRT1 and the oncogenic form of ␤-catenin in primary human tumors, with SIRT1 deacetylating ␤catenin and promoting its cytoplasmic localization (Firestein et al., 2008; Vlaicu et al., 2010).

(Vlaicu et al., 2010). Regarding CRC metastasis, a decreased level of H3K4me2 in the coding region of NDRG1 in SW620 cells was correlated with down-regulation of NDRG1 expression (Li and Chen, 2011). Another crucial histone mark of heterochromatin being associated with transcriptional repression is H3K9 tri-methylation. This was found increased in HCT116 cells, possibly promoting gene silencing of tumor suppressor genes (Nguyen et al., 2002). Similarly, overexpression of G9a, a H3K9-specific p53 methyltransferase, is reported in CRC among other types of cancer and it likely affects gene expression (Huang et al., 2010). Moreover, demethylation on the same residue is necessary for transcriptional activation of 15LOX-1, along with H3 and H4 acetylation reported earlier (Zuo et al., 2008).

5. Mechanisms of methylation marks in CRC 6. Regulatory role of histone methylation proteins in CRC Histone methylation takes place either on lysines or arginines. This dual residue targeting, along with multiple marks on the same residue such as mono-, di- and tri-methylation for lysines and mono- and di-methylation for arginines, lays extra complexity to the process (Kouzarides, 2007; Martin and Zhang, 2005). Enzymes mediating this modification are distinguished into lysine and arginine HMTs and lysine histone demethylases (HDMs). Functional complexity persists, since these methyl-modifications may have either an activating or a repressive role (Fig. 2), being recruited by different transcription factors and displaying enormous specificity compared to HATs (Bannister and Kouzarides, 2011; Lee et al., 2005; Fig. 1; Table 3). A classic histone methylation mark in CRC among other malignancies tends to be the loss of tri-methylation at lysine 20 of histone H4 (H4K20me3 ) in primary tumors and in cancer cell lines compared to normal mucosa, along with the global loss of DNA methylation and mono-acetylation at lysine 16, as previously described (Fraga et al., 2005). In other types of malignancy, this common hallmark has been detected in gene silencing processes during oncogenesis, being generally associated with repressed chromatin status (Kwon et al., 2010; Nishioka et al., 2002; Pogribny et al., 2006; Schotta et al., 2004). In addition, mono-, di- and tri-methylation of H3K4 seem to be marks of transcriptional activation and targets of SET and MYND domain-containing protein 3 (SMYD3) HMT and lysinespecific demethylase 1 (LSD1) HDM, both highly expressed in CRC (Hamamoto et al., 2004; Hayami et al., 2011). The first genomewide profiling data concerning histone methylation in CRC revealed similar profiles for H3K4me3 and H3K27me3 in normal colon and tumor tissues, differing only in CRC cell lines. This modification set by the Polycomb system is commonly associated with repressive chromatin domains (Francis et al., 2004; Kirmizis et al., 2004). Most important though, novel expression patterns came into sight with tumor genes that were positive for H3K4me3 already in normal tissue, becoming hyperactivated in tumors whereas genes with H3K27me3 and low expression in normal colon becoming hypersilenced in CRC tumors (Enroth et al., 2011). H3K4me3 and the loss of H3K27me3 along with increased H3 acetylation, were also associated with reactivation of silenced genes such as melanoma-associated antigen 3 (MAGEA3) and short-chain dehydrogenase/reductase family 42E mem (HSPC105) (Mossman and Scott, 2011) (Fig. 1). Additional DNA methylation in CRC-derived cell lines leads to reduced peroxisome proliferator-activated receptor gamma (PPAR) transcription due to recruitment of HDAC1, enhancer of zeste homolog 2 (EZH2) and methyl-CpG-binding protein 2 (MeCP2) (Pancione et al., 2010). H3K27me3 status was also found to decrease the promoter activity of cyclin D1 independently of the high methyltransferase enzyme level (Pelaez et al., 2010), and was further associated with the cell-cycle activator, RGC32 knockdown

6.1. Lysine HMTs HMTs may also acquire abnormal expression profiles and biological behavior linked to CRC oncogenic process (Ellis et al., 2009) (Table 4). The H3K4 myeloid/lymphoid leukemia (MLL) family of lysine HMTs is rather well studied due to the MLL1 protooncogene involved in myeloid and lymphoid leukemia (Krivtsov and Armstrong, 2007). A wide consensus coding sequence analysis revealed six somatic heterozygous mutations in MLL3 among 35 CRC tumors, two of them leading to truncation of the catalytic SET domain of the protein (Sjoblom et al., 2006). Likewise, another study showed that MLL3 was one of the most frequent deleted genes in African-American population compared to Caucasians with CRC, being affected of local chromosome shattering in primary and metastatic CRCs. Additionally, frameshift mutations in MLL3 seem to be more frequently associated with MSI (Ashktorab et al., 2010; Kloosterman et al., 2011; Watanabe et al., 2011a,b). However, contradictory data exist relative to the presence of MLL3 mutations in CRC tissues, further questioning the nascent role of this gene in colorectal carcinoma (Ahn et al., 2007; Vakoc et al., 2009). EZH2 represents an important lysine HMT grouped in the tumor suppressor gene silencers of the Polycomb group of proteins, targeting H3K27 methylation mark and interacting with several transcription factors (Cao et al., 2002; Kirmizis et al., 2004). In CRC, EZH2 overexpression is directly correlated with HDAC1 expression, being associated with worse prognosis of the patients (Mimori et al., 2005; Takawa et al., 2011; Wang et al., 2010; Crea et al., 2011). However, in a different study, increased EZH2 expression was associated with better cancer-free survival of CRC patients after surgery (Fluge et al., 2009). EZH2 down-regulation is further shown to prevent proliferation of CRC cells (Fussbroich et al., 2011). Correlation of protein levels with the transcription factor 12 (TCF12) suggests that the latter can suppress E-cadherin via complexes formed by the Polycomb group of proteins (Lee et al., 2012). EZH2 participates also in the PPAR silencing transcriptional machinery in CRC (Pancione et al., 2010), and it seems to subordinate DNA methylation in runtrelated transcription factor 3 (RUNX3) re-silencing after the removal of demethylating agents (Kodach et al., 2010). With regard to the PR domain zinc finger proteins (PRDM) family of lysine HMTs, the corresponding retinoblastoma proteininteracting zinc finger (RIZ1) gene (PRDM2) has been attributed a tumor suppressing role through targeting of H3K9 residue in various types of cancer (Steele-Perkins et al., 2001). In CRC, frequent frameshift mutations were detected in MSI primary tumors and CRC cell lines along with reduced mRNA expression of RIZ1 (Chadwick et al., 2000; Piao et al., 2000; Sakurada et al., 2001). RIZ1 protein expression is strictly located in MSI tumors and correlates

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1283

Table 3 Histone methylation marks in CRC. Methylation marks

Impaired function

Gene regulation

References

H3K4me1,2,3

Hypermethylation (CRC cell lines, CRC primary tumors)

Enroth et al. (2011) Hamamoto et al. (2004), Mossman and Scott (2011), Vlaicu et al. (2010)

Demethylation (CRC cell lines)

SMYD3 induced, Nkx2.8, C/EBP␦, Nkx2.5, Wnt10B, PIK3CB, NEURL, PSMD9, ECEL1, CRKL, APS and Seb4D activation, tumor-wide gene hyperactivation NDRG1 suppression

Hypermethylation (CRC cell lines)

PPAR, p14/p16, p53 silencing

Demethylation (CRC cell lines)

15-LOX-1 reactivation

Huang et al. (2010), Nguyen et al. (2002), Pancione et al. (2010) Zuo et al. (2008)

H3K27me

Hypermethylation (CRC primary tumors, CRC cell lines) Demethylation (CRC and Harvey-Ras CRC cell lines)

Tumor gene hypersilencing, PPAR silencing CDO1, HSPC105 and MAGEA3 reactivation, RGC32 silencing induced

H4K20me3

Demethylation (CRC primary tumors, CRC cell lines, other malignancies)

H3K9me3

3

significantly with female sex, proximal tumor location, stage B and grade III tumors (Emterling et al., 2004). RIZ1 presents a promising therapeutic target since its expression through adenovirus vectors was capable to inhibit tumor progression in MSI HCT116 CRC xenograft tumors and induce apoptosis of cancer cells more efficiently than those expressing p53 that were unable to induce tumor suppression (Jiang and Huang, 2001). SUV39H1, another H3K9 HMT, exhibits increased expression in CRC tumors being directly correlated with hypermethylation of certain gene promoters and significantly associated with DNA methyltransferase 1 (DNMT1) levels (Kang et al., 2007). Great interest, though, gathers SMYD3 HMT that is H3K4 specific and participates in the basal transcriptional machinery by forming a complex with RNA polymerase II through RNA helicase, HELZ. This interaction, along with tandem repeat polymorphisms in an E2F-binding element of the SMYD3 regulatory unit, allocates an oncogenic role to SMYD3. Its overexpression has been associated with increased risk for CRC development due to activation of oncogenes as well as homeobox and cell-cycle regulatory genes (Hamamoto et al., 2004; Tsuge et al., 2005; Xi et al., 2008). Additional evidence of SMYD3 correlation with CRC pathology includes overexpression of vascular endothelial growth factor receptor 1 (VEGFR1) as a SMYD3 methylation target and correlation of SMYD3 up-regulation with K-ras mutations (Gaedcke et al., 2010; Kunizaki

Li and Chen (2011)

Enroth et al. (2011) Li and Chen (2011), Pancione et al. (2010) Mossman and Scott (2011), Pelaez et al. (2010), Vlaicu et al. (2010) Fraga et al. (2005), Kwon et al. (2010), Nishioka et al. (2002), Pogribny et al. (2006), Schotta et al. (2004)

et al., 2007; Watanabe et al., 2011a,b). The HMT activity of SMYD3 is enhanced by a cleavage at the N-terminal part of the full-length protein present in CRC cells and other cancer cell lines (Silva et al., 2008). 6.2. Arginine HMTs and HDMs Data from arginine HMTs and their role in CRC are limited. Elucidation of the distribution pattern of protein arginine methyltransferase 1 (PRMT1) HMT in CRC primary tissues has revealed significant correlations between high PRMT1 expression and parameters of advanced malignancy as well as unfavorable prognosis (Mathioudaki et al., 2008; Papadokostopoulou et al., 2009). Protein arginine methyltransferase 5 (PRMT5) seems to interact with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in CRC cell lines and to inhibit apoptosis via NF-␬B (Tanaka et al., 2009). Co-activator-associated arginine methyltransferase 1 (CARM1), a transcriptional co-activator of androgen receptor signaling, is overexpressed in CRC as opposed to prostate and breast cancers (Kim et al., 2010). It is also suggested that CARM1 through specific methylation of H3R17 residue, regulates the activation of oncogenic Wnt pathway in RKO cells (Ou et al., 2011). Likewise, the role of HDMs is very little understood. As described earlier, LSD1, that reversely targets H3K4 and H3K9 residues, is

Table 4 HMTs and HDMs in CRC. Impaired function

References

Truncating mutations (MSI CRC primary tumors) Deletions (MSI CRC primary and metastatic tumors)

Sjoblom et al. (2006), Watanabe et al. (2011a,b) Ashktorab et al. (2010), Kloosterman et al. (2011)

EZH2

Overexpression (CRC primary tumors) Down-regulation (CRC cell lines) Mutation (CRC primary tumors)

Fluge et al. (2009), Mimori et al. (2005), Takawa et al. (2011) Fussbroich et al. (2011) Crea et al. (2011)

RIZ1

Frameshift mutations (MSI CRC primary tumors, CRC cell lines) Underexpression Overexpression (CRC primary tumors)

Chadwick et al. (2000), Piao et al. (2000), Sakurada et al. (2001)

Kang et al. (2007)

SMYD3

Overexpression (CRC primary tumors) Overexpression (CRC Kras mutations)

Tsuge et al. (2005), Xi et al. (2008) Gaedcke et al. (2010), Watanabe et al. (2011a,b)

PRMT1 CARM1

Overexpression (CRC primary tumors) Overexpression (CRC primary tumors)

Mathioudaki et al. (2008), Papadokostopoulou et al. (2009) Kim et al. (2010)

HDMs LSD1 JMJD1A

Overexpression (CRC primary tumors) Up-regulation (CRC cell lines)

Hayami et al. (2011) Uemura et al. (2010)

HMTs MLL3

SUV39H1

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significantly up-regulated in CRC primary tumors (Hayami et al., 2011). The largest HDM group, though, includes Jumonji C-domain containing histone demethylases (JHDMs). Unlike LSD1, JHDMs can demethylate mono-, di- and tri-methylated lysines. Specifically, Jumonji-domain containing 1A (JMJD1A) represents a biomarker of hypoxic tumor cells in CRC and its loss by small interfering RNA (siRNA) treatment was found to reduce proliferation and invasiveness in CRC cell lines. Furthermore, JMJD1A exhibited promising therapeutic potential in CRC xenografts (Uemura et al., 2010).

7. Epigenetic alterations interplay in CRC The ensemble of the histone modification mechanisms described do not act independently during CRC tumorigenesis, but are rather subjected to a dynamic crosstalk between other epigenetic events, such as DNA methylation and microRNAs (miRNAs). The continuous interaction of epigenetic mechanisms originates from the process that defines gene repression patterns during mammalian development, being mediated by biochemical interactions between HMT SET domain and DNMTs. More specifically, it is suggested that CRC and other cancer cell types are liable to a preprogrammed pathologic de novo methylation, which occurs at gene promoter sites – including tumor suppressor genes and other genes – previously modified by proteins of the Polycomb group at an early stage of oncogenesis (Cedar and Bergman, 2009; Keshet et al., 2006) (Fig. 3A). This emerging concept rules out the theories for “selection” processes that were believed in preliminary studies (Jones and Baylin, 2007). The Polycomb protein group being associated with heterochromatin transcription repressive status is divided into two subgroups, depending on the proteins that participate in the multimeric complexes (Sparmann and van Lohuizen, 2006). EZH2, along with other proteins such as embryonic ectoderm development protein (EED) and suppressor of zeste 12 homolog (SUZ12), form the Polycomb repressive complex 2 (PRC2) in order to maintain the H3K27me3 inactivating mark (Kirmizis et al., 2004; Richly et al., 2011). This specific repressive mark is suggested to be directly associated with methylated CpG islands gene sites in CRC cells, detected not only in embryonic stem cells but also in differentiated colon tissue (Rada-Iglesias et al., 2009). The overall suggestion sets de novo methylation as a process that is already targeted by preexisting histone methylation marks of the Polycomb protein group, silencing genes that inhibit proliferation in CRC, in particular cell adhesion inducers and Wnt pathway antagonists (Schlesinger et al., 2007). The next arising question is whether the presence of histone modifications participates in triggering the onset of CpG islands de novo methylation in CRC, apart of specific targeting DNA methylation through Polycomb. This seems possible given that EZH2 has been found to be a prerequisite for DNA methylation establishment by altering its expression levels and directly interacting with DNMTs (Varambally et al., 2002; Vire et al., 2006). In CRC, DNA methyltransferase 3B (DNMT3B) presents an important enzyme that links to Polycomb repressive complex 1 (PRC1) mark in silenced genes, with nearly 50% of DNMT3B regulated genes being also epigenetically modulated by PRC1 or PRC2 (Jin et al., 2009). A third epigenetic player enters the game, as it is suggested that functional elevation of the interacting factors may be regulated by microRNAs (miRNAs) (Sinkkonen et al., 2008). miRNAs represent short non-coding RNAs (ncRNAs) of approximately 22 nucleotides constituting an additional regulatory mechanism of mRNA expression. They bind specific sequences of certain target genes, leading to inhibition of translation and mRNA itself. A large volume of data attribute to miRNAs either an oncogenic or tumor suppressing role (Schnekenburger and Diederich, 2012). Besides modulation of DNA methylation potential, miRNAs have been implicated in

crucial pathways engaged in CRC formation, such as Wnt and p53 signaling, apoptosis, differentiation and proliferation pathways, as well as the processes of invasion and metastasis. Regarding the regulation of epigenetic mechanisms in CRC, the long ncRNA Hox transcript antisense intergenic RNA (HOTAIR) acquires increased expression in tumor tissue that correlates with liver metastases and poor prognosis, but also with the PRC2 factors SUZ12, EZH2 and H3K27me3 further confirming similar findings in breast cancer (Kogo et al., 2011). Moreover, Dicer, a siRNA/miRNA enzyme known in gene silencing mechanisms of yeast and plants, is essential for DNA hypermethylation–transcriptional repression establishment of certain genes in CRC cells (Ting et al., 2008). Recently, protein-coding RNA transcripts possessing miRNA response elements (MREs) have been shown to compete for binding to common miRNAs and are thus termed competitive endogenous RNAs (ceRNAs). Consequently, ceRNAs seem to be part of a unified hypothesis, which poses that mRNAs, transcribed pseudogenes (non-functional genomic loci) and long ncRNAs that bear MREs participate in interregulatory genetic networks, through a continuous crosstalk, thus altering cellular homeostasis. ncRNAs with ceRNAs may function as strong tumor suppressing or oncogenic candidates, hence generating a new cancer-related research field cancer with promising experimental data (Salmena et al., 2011). The recent identification of tumor suppressive phosphatase and tensin homolog (PTEN) regulatory ceRNAs (ZEB2) in a mouse model of melanoma and its implication in tumorigenic process, provides further verification of this hypothesis (Karreth et al., 2011; Tay et al., 2011). Following DNA methylation, histone marks that originally initiated the process seem to be withdrawn and rather allow DNMT1 to take turn in order to maintain gene silencing and respective transcriptionally repressed chromatin status. This is evident in CRC and other tumor cell types where DNA methylation remains still after EZH2 depletion (McGarvey et al., 2007). It seems as though DNA methylation is the dominant repressive mark that partially replaces the previous histone methylated heterochromatin status. Several studies in CRC cell lines also suggest that gene reactivation needs demethylation at first and, then, histone acetylation in order to occur, as in the case of MLH1, tissue inhibitor of metalloproteinase 3 (TIMP3) and p16INK4A (Bachman et al., 2003; Cameron et al., 1999). The DNA methylation-histone acetylation interplay is mediated via methyl-CpG-binding domain proteins (MBDs) that recruit HDACs and interact with them in order to form hetrerochromatin structures, thus providing an additional mechanism of imposing gene silencing (Jones et al., 1998; Nan et al., 1998) (Fig. 3B). Members of this family include MBD1, 2, 3, MeCP2 and KAISO that have been associated with transcriptional repression. In CRC cell lines, MBD2 binds to methyl-CpG regions of p16 and p14 cell-cycle genes promoting their silencing probably through HDAC recruitment (Magdinier and Wolffe, 2001; Rountree et al., 2000; Sun et al., 2012). In addition, DNMT1 forms a direct complex with HDAC2 in CRC cells along with a novel protein, DNA methyltransferase 1associated protein 1 (DMAP1), in order to repress transcription in late S-phase of the cell cycle (Rountree et al., 2000).

8. Regulation of histone modifications in clinical practice Histone dynamic alterations during the process of carcinogenesis increasingly emerge as mechanisms of great significance. Their reversible nature renders them prominent targets for pharmaceutical therapy and a number of clinical trials focused on several enzymes have been reported. The most thoroughly investigated agents are HDAC inhibitors (HDACi) (Bolden et al., 2006). HDACi can effectively suppress HDAC enzymatic activity of classes I and II confirming their important role in colon cell proliferation and differentiation (Mariadason, 2008a). Short-chain fatty acids

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Fig. 3. Examples of epigenetic interplay leading to colorectal cancer (CRC) pathology. (A) EZH2 (orange) along with EED (light orange) and SUZ12 (yellow) form the Polycomb complex, PRC2 (black) which recruits PRC1 to induce elevation of H3K27me3 repressive mark (dark red cross). Up-regulation of EZH2 expression is required for establishment of DNA CpG islands methylation, mediated by DNMT3B (green), an important regulatory enzyme in CRC. This process targets mostly (by suppressing) cell adhesion molecules and Wnt antagonists. (B) Methylated CpG islands attract MBDs (yellow) which further recruit HDACs (red) in order to repress tumor suppressor genes. DNMT1 (green) forms a complex with HDAC2 (red) and DMAP1 protein (blue) that has been associated with repression of CRC suppressor gene transcription. Ac, acetylation; EZH2, enhancer of zeste homolog 2; EED, embryonic ectoderm development protein; DMAP1, DNA methyltransferase 1-associated protein 1; DNMT1 and 3B, DNA methyltransferase 1 and 3B; HDACs and HDAC2, histone deacetylases and histone deacetylase 2; K, lysine; MBDs, methyl-CpG-binding domain proteins; Me, methylation; PRC1 and 2, Polycomb repressive complex 1 and 2; SUZ12, suppressor of zeste 12 homolog.

and butyrate have been characterized and utilized for decades (Riggs et al., 1977), while electrophilic ketones (trifluoromethylketone), benzamides (MS-275), cyclic tetrapeptides (depsipeptide) and hydroxamic acids (TSA and suberoylanilide hydroxamic acid [SAHA]) also fill in the group. HDACi exert their anti-tumor effects in CRC cells either by preventing cell proliferation or by inducing differentiation and apoptosis (Hague et al., 1993; Heerdt et al., 1994; Kim et al., 1980) (Fig. 4). Similar effects have been documented in vivo since certain HDACi can induce tumor growth decline in CRC rat models (Wong et al., 2005). The key protein that seems to mediate HDACi effect on growth arrest is p21, which is induced directly and rapidly in a Sp1/Sp3-dependent manner and independently of p53 (Archer et al., 1998; Nakano et al., 1997). HDACi also act on a number of other proteins related to growth inhibition, such as p16, and can repress several proto-oncogenes, such as c-myc, without adequate elucidation of the underlying mechanisms (Schwartz et al., 1998; Wilson et al., 2002). As previously described, HDACs are mostly overexpressed in CRC primary tumors by introducing hypoacetylated inactivating transcription marks. Consequently, HDACi likely exert their action through transcriptional regulation by reversing repressive acetylation marks on tumor suppressor genes, thus inducing re-expression of certain gatekeeper genes involved in proliferation and physiological growth status (Bolden et al., 2006) (Fig. 4). However, HDACs interact and acetylate also non-histone proteins. HDACi effects may also result from a second mechanism that promotes acetylation of non-histone proteins, altering their transcriptional activity (Johnstone and Licht, 2003). They can potentially hyperacetylate transcription factors, such as NF-␬B, E2F1, p53, signal transducer

Fig. 4. Targeting mechanisms employed by HDAC inhibitors (HDACi). HDACi induce differentiation and apoptosis of colorectal cancer (CRC) cells while inhibit their proliferation by four potential mechanisms. TFs, transcription factors.

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and activator of transcription 1 and 3 (STAT1, STAT3), which regulate gene expression in favor of growth arrest and apoptosis (Chen et al., 2001; Gu and Roeder, 1997; Martinez-Balbas et al., 2000; Yuan et al., 2005). A third regulatory mechanism attributed to HDACi may include hyperacetylation of cytoplasmic proteins, thus inhibiting their oncogenic activity via transcription-independent processes (Mariadason, 2008a) (Fig. 4). An additional fourth mechanism assigns HDACi a potential regulatory role over the immune response toward tumor progression and neovascularization. HDACi can either modulate and/or make cancer cells more prominent to targeting by immune system, or even alter the cell’s own immune activity and cytokine production (Bolden et al., 2006) (Fig. 4). A significant number of HDACi is being already evaluated in clinical trials of cancer patients (Peterson and Laniel, 2004). Specifically in CRC, the HDACi SAHA has been tested in combination with classic chemotherapeutic drugs, 5FU/Leucovorin (LV) and FOLFOX (5FU/LV/oxaliplatin), a combination that has proved promising in xenograft models (Tumber et al., 2007). A synergistic effect of HDACi with radiation has also been suggested, partially being mediated by hyperacetylation of histone residues that creates increased access of radiation to DNA (Karagiannis and El-Osta, 2006). Along with the development of new histone-related anti-tumor agents, the synergistic effect of DNA demethylation compounds and HDACi is currently being tested. Although their independent use has profound effects in CRC, their combination also shows significant results concerning silenced gene reactivation and inhibition of cell proliferation (Glaser, 2007). A synergistic effect was demonstrated when epigenetic agents were tested along with signal transduction inhibitors (Bolden et al., 2006). The combination of HDACi with other targeted therapies is of great interest and the underpinning mechanisms still remain to be elucidated, especially relative to CRC treatment that is characterized by a plethora of genetic and epigenetic aberrations.

9. Conclusions and future prospects CRC follows an ultimate genetic model, in which a significant amount of knowledge has been acquired in favor of an overall approach of cancer biology, during the last years. Current epigenetic data elaborate further this model providing new insights, additional complexity and better understanding of the oncogenic process. Aberrant post-translational histone modifications play a key role in this model, linking the function of other epigenetic categories, but also elucidating further aspects of impaired gene regulation. Although aberrant DNA methylation is the best characterized epigenetic event associated with tumor biology so far, histone modifications emerge as crucial co-factors. Functional clarification of histone-modifying proteins relative to their impact on specific histone marks that define heterochromatin and euchromatin status, has reached a satisfying level of understanding mainly focusing on tumor suppressor gene silencing. As aforementioned, several key histone enzymes emerge as novel prognostic biomarkers in CRC, providing already a useful prognostic tool (Fraga et al., 2005; Giannini and Cavallini, 2005; Gorrini et al., 2007; Huang et al., 2005; Ishihama et al., 2007; Sakuraba et al., 2009). While this knowledge establishes tissue-specific expression patterns and biological behavior of HATs, HDACs, HMTs and HDMs, contradictory studies exist regarding the specific role and mechanism of action of each of these molecules. Experimental data suggest that parallel mechanisms (i.e. interplay with DNA methylation and interaction with non-histone proteins) take place which need to be taken into consideration as a whole for proper management of CRC hallmark events (Hanahan and Weinberg, 2011). Histone

modifications are engaged in tumor invasion and metastasis, angiogenesis, self-sufficiency in growth signals, insensitivity to growth inhibitors and limitless replicative potential. In addition to interplay of DNA methylation and histone modifications, the potential role of non-histone substrates modification emerges in regulation of gene expression, with particular emphasis on the acetylation and methylation of transcription factors. The acetylation of several transcription factors related to CRC oncogenesis, such as NF-␬B and p53, along with the capability of HATs for automodification resembling kinases function, attributes acetylation a wider impact to signal transduction similar to phosphorylation (Bannister and Miska, 2000). The reversible methylation of crucial oncogenic promoter-bound transcription factors has also been suggested by histone-modifying enzymes. Specifically, mutations and deregulated activity of lysine methyltransferases and demethylases may enhance tumorigenesis through alternative transcription factorrelated mechanisms (Stark et al., 2011). It is clear that these covalent modifications go beyond histone modifications and chromatin regulation. Future research is guided toward two main directions: the elucidation of the epigenetic cascade of events that characterize CRC and cancer in general, and most importantly, the identification of the ways that acetylation and methylation modulate protein–protein interactions integrating their impact on transcription factor regulation. Current therapeutic approaches are directed to either smallmolecule inhibitors targeting the enzymes that mediate chromatin modifications, or toward chemopreventive agents (such as natural compounds) targeting global epigenetic deregulations. DNMT and HDAC inhibitors, such as decitabine and SAHA, have been effectively applied in various cancer types along with CRC, exhibiting pleiotropic effects that include regulation of cell growth and differentiation, senescence and apoptosis, tumor-cell sensitization to chemotherapeutic agents, or acting synergistically with anti-tumor compounds (Schnekenburger and Diederich, 2012). However, they have been associated with non-specific cytotoxic effects and dosedependent toxicities including thrombocytopenia and neutropenia, further restricting their routine clinical use. At this point dietary agents, natural molecules and nutrients have been applied as a more safe epigenetic modulation approach. Although in vitro data point toward an extensive list of chemopreventive agents with global epigenetic effects (micronutrients, antioxidants, polyphenols, soy isoflavones, lycopene, sulfur-containing compounds of cruciferous vegetables, resveratrol and others), their functional effectiveness and relevance to prevention, early cancer detection and effective chemoprevention requires further investigation (Huang et al., 2011). Studies focused on the elucidation of the detailed mechanism of action of these dietary modulators along with safe dose range and efficiency in humans is highly demanded. Future therapeutic approaches should be directed toward identification of efficient strategies for chemopreventive intervention, taking into consideration the importance of epigenetic mechanisms in regulating gene expression outcomes.

References Ahn CH, Yoo NJ, Lee JW, Lee SH. Absence of MLL3 mutations in colorectal carcinomas of Korean patients. APMIS 2007;115:859–60. Archer SY, Meng S, Shei A, Hodin RA. p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells. Proceedings of the National Academy of Sciences of the United States of America 1998;95:6791–6. Ashktorab H, Belgrave K, Hosseinkhah F, Brim H, Nouraie M, Takkikto M, et al. Global histone H4 acetylation and HDAC2 expression in colon adenoma and carcinoma. Digestive Diseases and Sciences 2009;54:2109–17. Ashktorab H, Schaffer AA, Daremipouran M, Smoot DT, Lee E, Brim H. Distinct genetic alterations in colorectal cancer. PLoS One 2010;5:e8879. Avvakumov N, Cote J. The MYST family of histone acetyltransferases and their intimate links to cancer. Oncogene 2007;26:5395–407.

A.N. Gargalionis et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1276–1289 Bachman KE, Park BH, Rhee I, Rajagopalan H, Herman JG, Baylin SB, et al. Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene. Cancer Cell 2003;3:89–95. Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell Research 2011;21:381–95. Bannister AJ, Miska EA. Regulation of gene expression by transcription factor acetylation. Cellular and Molecular Life Sciences 2000;57:1184–92. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nature Reviews Drug Discovery 2006;5:769–84. Brady ME, Ozanne DM, Gaughan L, Waite I, Cook S, Neal DE, et al. Tip60 is a nuclear hormone receptor coactivator. Journal of Biological Chemistry 1999;274:17599–604. Bryan EJ, Jokubaitis VJ, Chamberlain NL, Baxter SW, Dawson E, Choong DY, et al. Mutation analysis of EP300 in colon, breast and ovarian carcinomas. International Journal of Cancer 2002;102:137–41. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nature Genetics 1999;21:103–7. Campbell IG, Choong D, Chenevix-Trench G. No germline mutations in the histone acetyltransferase gene EP300 in BRCA1 and BRCA2 negative families with breast cancer and gastric, pancreatic, or colorectal cancer. Breast Cancer Research 2004;6:R366–71. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, et al. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 2002;298:1039–43. Cedar H, Bergman Y. Linking DNA methylation and histone modification: patterns and paradigms. Nature Reviews Genetics 2009;10:295–304. Chadwick RB, Jiang GL, Bennington GA, Yuan B, Johnson CK, Stevens MW, et al. Candidate tumor suppressor RIZ is frequently involved in colorectal carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America 2000;97:2662–7. Chen L, Fischle W, Verdin E, Greene WC. Duration of nuclear NF-␬B action regulated by reversible acetylation. Science 2001;293:1653–7. Chen YX, Fang JY, Lu R, Qiu DK. Expression of p21(WAF1) is related to acetylation of histone H3 in total chromatin in human colorectal cancer. World Journal of Gastroenterology 2007;13:2209–13. Cosgrove MS, Boeke JD, Wolberger C. Regulated nucleosome mobility and the histone code. Nature Structural & Molecular Biology 2004;11:1037–43. Crea F, Fornaro L, Paolicchi E, Masi G, Frumento P, Loupakis F, et al. An EZH2 polymorphism is associated with clinical outcome in metastatic colorectal cancer patients. Annals of Oncology 2011 (Epub ahead of print). Davis PK, Brackmann RK. Chromatin remodeling and cancer. Cancer Biology and Therapy 2003;2:22–9. Ellis L, Atadja PW, Johnstone RW. Epigenetics in cancer: targeting chromatin modifications. Molecular Cancer Therapeutics 2009;8:1409–20. Emterling A, Wallin A, Arbman G, Sun XF. Clinicopathological significance of microsatellite instability and mutated RIZ in colorectal cancer. Annals of Oncology 2004;15:242–6. Enroth S, Rada-Iglesisas A, Andersson R, Wallerman O, Wanders A, Pahlman L, et al. Cancer associated epigenetic transitions identified by genome-wide histone methylation binding profiles in human colorectal cancer samples and paired normal mucosa. BMC Cancer 2011;11:450. Evans PM, Zhang W, Chen X, Yang J, Bhakat KK, Liu C. Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation. Journal of Biological Chemistry 2007;282:33994–4002. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759–67. Federico M, Bagella L. Histone deacetylase inhibitors in the treatment of hematological malignancies and solid tumors. Journal of Biomedicine and Biotechnology 2011;2011:475641. Firestein R, Blander G, Michan S, Oberdoerffer P, Ogino S, Campbell J, et al. The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth. PLoS One 2008;3:e2020. Fluge O, Gravdal K, Carlsen E, Vonen B, Kjellevold K, Refsum S, et al. Expression of EZH2 and Ki-67 in colorectal cancer and associations with treatment response and prognosis. British Journal of Cancer 2009;101:1282–9. Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nature Genetics 2005;37:391–400. Francis NJ, Kingston RE, Woodcock CL. Chromatin compaction by a polycomb group protein complex. Science 2004;306:1574–7. Frank SR, Parisi T, Taubert S, Fernandez P, Fuchs M, Chan HM, et al. MYC recruits the TIP60 histone acetyltransferase complex to chromatin. EMBO Reports 2003;4:575–80. Fullgrabe J, Kavanagh E, Joseph B. Histone onco-modifications. Oncogene 2011;30:3391–403. Fussbroich B, Wagener N, Macher-Goeppinger S, Benner A, Falth M, Sultmann H, et al. EZH2 depletion blocks the proliferation of colon cancer cells. PLoS One 2011;6:e21651. Gaedcke J, Grade M, Jung K, Camps J, Jo P, Emons G, et al. Mutated KRAS results in overexpression of DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase, in rectal carcinomas. Genes, Chromosomes and Cancer 2010;49:1024–34. Gaughan L, Brady ME, Cook S, Neal DE, Robson CN. Tip60 is a co-activator specific for class I nuclear hormone receptors. Journal of Biological Chemistry 2001;276:46841–8.

1287

Gaughan L, Logan IR, Cook S, Neal DE, Robson CN. Tip60 and histone deacetylase 1 regulate androgen receptor activity through changes to the acetylation status of the receptor. Journal of Biological Chemistry 2002;277:25904–13. Gayther SA, Batley SJ, Linger L, Bannister A, Thorpe K, Chin SF, et al. Mutations truncating the EP300 acetylase in human cancers. Nature Genetics 2000;24:300–3. Ge X, Jin Q, Zhang F, Yan T, Zhai Q. PCAF acetylates {beta}-catenin and improves its stability. Molecular Biology of the Cell 2009;20:419–27. Giannini R, Cavallini A. Expression analysis of a subset of coregulators and three nuclear receptors in human colorectal carcinoma. Anticancer Research 2005;25:4287–92. Glaser KB. HDAC inhibitors: clinical update and mechanism-based potential. Biochemical Pharmacology 2007;74:659–71. Godman CA, Joshi R, Tierney BR, Greenspan E, Rasmussen TP, Wang HW, et al. HDAC3 impacts multiple oncogenic pathways in colon cancer cells with effects on Wnt and vitamin D signaling. Cancer Biology and Therapy 2008;7:1570–80. Gorrini C, Squatrito M, Luise C, Syed N, Perna D, Wark L, et al. Tip60 is a haploinsufficient tumour suppressor required for an oncogene-induced DNA damage response. Nature 2007;448:1063–7. Grunstein M. Histone acetylation in chromatin structure and transcription. Nature 1997;389:349–52. Gu W, Roeder RG. Activation of p53sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997;90:595–606. Hague A, Manning AM, Hanlon KA, Huschtscha LI, Hart D, Paraskeva C. Sodium butyrate induces apoptosis in human colonic tumour cell lines in a p53independent pathway: implications for the possible role of dietary fibre in the prevention of large-bowel cancer. International Journal of Cancer 1993;55:498–505. Hamamoto R, Furukawa Y, Morita M, Iimura Y, Silva FP, Li M, et al. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nature Cell Biology 2004;6:731–40. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74. Hanigan CL, Van Engeland M, De Bruine AP, Wouters KA, Weijenberg MP, Eshleman JR, et al. An inactivating mutation in HDAC2 leads to dysregulation of apoptosis mediated by APAF1. Gastroenterology 2008;135:1654–64, e2. Hayami S, Kelly JD, Cho HS, Yoshimatsu M, Unoki M, Tsunoda T, et al. Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers. International Journal of Cancer 2011;128:574–86. Heerdt BG, Houston MA, Augenlicht LH. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines. Cancer Research 1994;54:3288–93. Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JP, et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proceedings of the National Academy of Sciences of the United States of America 1998;95:6870–5. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. New England Journal of Medicine 2003;349:2042–54. Higashijima J, Kurita N, Miyatani T, Yoshikawa K, Morimoto S, Nishioka M, et al. Expression of histone deacetylase 1 and metastasis-associated protein 1 as prognostic factors in colon cancer. Oncology Reports 2011;26:343–8. Huang BH, Laban M, Leung CH, Lee L, Lee CK, Salto-Tellez M, et al. Inhibition of histone deacetylase 2 increases apoptosis and p21Cip1/WAF1 expression, independent of histone deacetylase 1. Cell Death and Differentiation 2005;12:395–404. Huang J, Dorsey J, Chuikov S, Perez-Burgos L, Zhang X, Jenuwein T, et al. G9a and Glp methylate lysine 373 in the tumor suppressor p53. Journal of Biological Chemistry 2010;285:9636–41. Huang J, Plass C, Gerhauser C. Cancer chemoprevention by targeting the epigenome. Current Drug Targets 2011;12:1925–56. Ionov Y, Matsui S, Cowell JK. A role for p300/CREB binding protein genes in promoting cancer progression in colon cancer cell lines with microsatellite instability. Proceedings of the National Academy of Sciences of the United States of America 2004;101:1273–8. Ishihama K, Yamakawa M, Semba S, Takeda H, Kawata S, Kimura S, et al. Expression of HDAC1 and CBP/p300 in human colorectal carcinomas. Journal of Clinical Pathology 2007;60:1205–10. Iyer NG, Ozdag H, Caldas C. p300/CBP and cancer. Oncogene 2004;23:4225–31. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global Cancer Statistics. CA: A Cancer Journal for Clinicians 2011;61:69–90. Jiang GL, Huang S. Adenovirus expressing RIZ1 in tumor suppressor gene therapy of microsatellite-unstable colorectal cancers. Cancer Research 2001;61:1796–8. Jin B, Yao B, Li JL, Fields CR, Delmas AL, Liu C, et al. DNMT1 and DNMT3B modulate distinct polycomb-mediated histone modifications in colon cancer. Cancer Research 2009;69:7412–21. Johnstone RW, Licht JD. Histone deacetylase inhibitors in cancer therapy: is transcription the primary target. Cancer Cell 2003;4:13–8. Jones PA, Baylin SB. The epigenomics of cancer. Cell 2007;128:683–92. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nature Genetics 1998;19:187–91. Kabra N, Li Z, Chen L, Li B, Zhang X, Wang C, et al. SirT1 is an inhibitor of proliferation and tumor formation in colon cancer. Journal of Biological Chemistry 2009;284:18210–7. Kang MY, Lee BB, Kim YH, Chang DK, Kyu Park S, Chun HK, et al. Association of the SUV39H1 histone methyltransferase with the DNA methyltransferase 1 at mRNA expression level in primary colorectal cancer. International Journal of Cancer 2007;121:2192–7.

1288

A.N. Gargalionis et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1276–1289

Karagiannis TC, El-Osta A. Modulation of cellular radiation responses by histone deacetylase inhibitors. Oncogene 2006;25:3885–93. Karreth FA, Tay Y, Perna D, Ala U, Tan SM, Rust AG, et al. In vivo identification of tumor-suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse model of melanoma. Cell 2011;147:382–95. Keshet I, Schlesinger Y, Farkash S, Rand E, Hecht M, Segal E, et al. Evidence for an instructive mechanism of de novo methylation in cancer cells. Nature Genetics 2006;38:149–53. Kim YR, Lee BK, Park RY, Nguyen NT, Bae JA, Kwon DD, et al. Differential CARM1 expression in prostate and colorectal cancers. BMC Cancer 2010;10:197. Kim YS, Tsao D, Siddiqui B, Whitehead JS, Arnstein P, Bennett J, et al. Effects of sodium butyrate and dimethylsulfoxide on biochemical properties of human colon cancer cells. Cancer 1980;45:1185–92. Kirmizis A, Bartley SM, Kuzmichev A, Margueron R, Reinberg D, Green R, et al. Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27. Genes and Development 2004;18:1592–605. Kloosterman WP, Hoogstraat M, Paling O, Tavakoli-Yaraki M, Renkens I, Vermaat JS, et al. Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer. Genome Biology 2011;12:R103. Knudson AG. Two genetic hits (more or less) to cancer. Nature Reviews Cancer 2001;1:157–62. Kodach LL, Jacobs RJ, Heijmans J, van Noesel CJ, Langers AM, Verspaget HW, et al. The role of EZH2 and DNA methylation in the silencing of the tumour suppressor RUNX3 in colorectal cancer. Carcinogenesis 2010;31:1567–75. Kogo R, Shimamura T, Mimori K, Kawahara K, Imoto S, Sudo T, et al. Long noncoding RNA HOTAIR regulates polycomb-dependent chromatin modification and is associated with poor prognosis in colorectal cancers. Cancer Research 2011;71:6320–6. Konishi K, Issa JP. Targeting aberrant chromatin structure in colorectal carcinomas. Cancer Journal 2007;13:49–55. Kouzarides T. Chromatin modifications and their function. Cell 2007;128:693–705. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nature Reviews Cancer 2007;7:823–33. Kunizaki M, Hamamoto R, Silva FP, Yamaguchi K, Nagayasu T, Shibuya M, et al. The lysine 831 of vascular endothelial growth factor receptor 1 is a novel target of methylation by SMYD3. Cancer Research 2007;67:10759–65. Kuzmichev A, Margueron R, Vaquero A, Preissner TS, Scher M, Kirmizis A, et al. Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation. Proceedings of the National Academy of Sciences of the United States of America 2005;102:1859–64. Kwon MJ, Kim SS, Choi YL, Jung HS, Balch C, Kim SH, et al. Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications. Carcinogenesis 2010;31:974–83. Lee CC, Chen WS, Chen CC, Chen LL, Lin YS, Fan CS, et al. TCF12 protein functions as transcriptional repressor of E-cadherin, and its overexpression is correlated with metastasis of colorectal cancer. Journal of Biological Chemistry 2012;287:2798–809. Lee DY, Teyssier C, Strahl BD, Stallcup MR. Role of protein methylation in regulation of transcription. Endocrine Reviews 2005;26:147–70. Li Q, Chen H. Transcriptional silencing of N-Myc downstream-regulated gene 1 (NDRG1) in metastatic colon cancer cell line SW620. Clinical and Experimental Metastasis 2011;28:127–35. Liu Y, Hong Y, Zhao Y, Ismail TM, Wong Y, Eu KW, et al. Histone H3 (lys-9) deacetylation is associated with transcriptional silencing of E-cadherin in colorectal cancer cell lines. Cancer Investigation 2008;26:575–82. Lleonart ME, Vidal F, Gallardo D, Diaz-Fuertes M, Rojo F, Cuatrecasas M, et al. New p53 related genes in human tumors: significant downregulation in colon and lung carcinomas. Oncology Reports 2006;16:603–8. Magdinier F, Wolffe AP. Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia. Proceedings of the National Academy of Sciences of the United States of America 2001;98:4990–5. Mariadason JM. HDACs and HDAC inhibitors in colon cancer. Epigenetics 2008a;3:28–37. Mariadason JM. Making sense of HDAC2 mutations in colon cancer. Gastroenterology 2008b;135:1457–9. Martin C, Zhang Y. The diverse functions of histone lysine methylation. Nature Reviews Molecular Cell Biology 2005;6:838–49. Martinez-Balbas MA, Bauer UM, Nielsen SJ, Brehm A, Kouzarides T. Regulation of E2F1 activity by acetylation. EMBO Journal 2000;19:662–71. Mathioudaki K, Papadokostopoulou A, Scorilas A, Xynopoulos D, Agnanti N, Talieri M. The PRMT1 gene expression pattern in colon cancer. British Journal of Cancer 2008;99:2094–9. Mattera L, Escaffit F, Pillaire MJ, Selves J, Tyteca S, Hoffmann JS, et al. The p400/Tip60 ratio is critical for colorectal cancer cell proliferation through DNA damage response pathways. Oncogene 2009;28:1506–17. McGarvey KM, Greene E, Fahrner JA, Jenuwein T, Baylin SB. DNA methylation and complete transcriptional silencing of cancer genes persist after depletion of EZH2. Cancer Research 2007;67:5097–102. Mimori K, Ogawa K, Okamoto M, Sudo T, Inoue H, Mori M. Clinical significance of enhancer of zeste homolog 2 expression in colorectal cancer cases. European Journal of Surgical Oncology 2005;31:376–80. Miremadi A, Oestergaard MZ, Pharoah PD, Caldas C. Cancer genetics of epigenetic genes. Human Molecular Genetics 2007;16(Spec No. 1):R28–49. Mossman D, Scott RJ. Long term transcriptional reactivation of epigenetically silenced genes in colorectal cancer cells requires DNA hypomethylation and histone acetylation. PLoS One 2011;6:e23127.

Muraoka M, Konishi M, Kikuchi-Yanoshita R, Tanaka K, Shitara N, Chong JM, et al. p300 gene alterations in colorectal and gastric carcinomas. Oncogene 1996;12:1565–9. Nakagawa M, Oda Y, Eguchi T, Aishima S, Yao T, Hosoi F, et al. Expression profile of class I histone deacetylases in human cancer tissues. Oncology Reports 2007;18:769–74. Nakano K, Mizuno T, Sowa Y, Orita T, Yoshino T, Okuyama Y, et al. Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line. Journal of Biological Chemistry 1997;272:22199–206. Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 1998;393:386–9. Nguyen CT, Weisenberger DJ, Velicescu M, Gonzales FA, Lin JC, Liang G, et al. Histone H3-lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-aza-2 -deoxycytidine. Cancer Research 2002;62:6456–61. Nishioka K, Rice JC, Sarma K, Erdjument-Bromage H, Werner J, Wang Y, et al. PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. Molecular Cell 2002;9:1201–13. Nosho K, Shima K, Irahara N, Kure S, Firestein R, Baba Y, et al. SIRT1 histone deacetylase expression is associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Modern Pathology 2009;22:922–32. Ou CY, LaBonte MJ, Manegold PC, So AY, Ianculescu I, Gerke DS, et al. A coactivator role of CARM1 in the dysregulation of beta-catenin activity in colorectal cancer cell growth and gene expression. Molecular Cancer Research 2011;9:660–70. Ozdag H, Batley SJ, Forsti A, Iyer NG, Daigo Y, Boutell J, et al. Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours. British Journal of Cancer 2002;87:1162–5. Ozdag H, Teschendorff AE, Ahmed AA, Hyland SJ, Blenkiron C, Bobrow L, et al. Differential expression of selected histone modifier genes in human solid cancers. BMC Genomics 2006;7:90. Pancione M, Sabatino L, Fucci A, Carafa V, Nebbioso A, Forte N, et al. Epigenetic silencing of peroxisome proliferator-activated receptor gamma is a biomarker for colorectal cancer progression and adverse patients’ outcome. PLoS One 2010;5:e14229. Papadokostopoulou A, Mathioudaki K, Scorilas A, Xynopoulos D, Ardavanis A, Kouroumalis E, et al. Colon cancer and protein arginine methyltransferase 1 gene expression. Anticancer Research 2009;29:1361–6. Pelaez IM, Kalogeropoulou M, Ferraro A, Voulgari A, Pankotai T, Boros I, et al. Oncogenic RAS alters the global and gene-specific histone modification pattern during epithelial-mesenchymal transition in colorectal carcinoma cells. International Journal of Biochemistry and Cell Biology 2010;42:911–20. Pena C, Garcia JM, Garcia V, Silva J, Dominguez G, Rodriguez R, et al. The expression levels of the transcriptional regulators p300 and CtBP modulate the correlations between SNAIL, ZEB1, E-cadherin and vitamin D receptor in human colon carcinomas. International Journal of Cancer 2006;119:2098–104. Peterson CL, Laniel MA. Histones and histone modifications. Current Biology 2004;14:R546–51. Piao Z, Fang W, Malkhosyan S, Kim H, Horii A, Perucho M, et al. Frequent frameshift mutations of RIZ in sporadic gastrointestinal and endometrial carcinomas with microsatellite instability. Cancer Research 2000;60:4701–4. Pogribny IP, Ross SA, Tryndyak VP, Pogribna M, Poirier LA, Karpinets TV. Histone H3 lysine 9 and H4 lysine 20 trimethylation and the expression of Suv4-20h2 and Suv-39h1 histone methyltransferases in hepatocarcinogenesis induced by methyl deficiency in rats. Carcinogenesis 2006;27:1180–6. Pruitt K, Zinn RL, Ohm JE, McGarvey KM, Kang SH, Watkins DN, et al. Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation. PLoS Genetics 2006;2:e40. Rada-Iglesias A, Enroth S, Andersson R, Wanders A, Pahlman L, Komorowski J, et al. Histone H3 lysine 27 trimethylation in adult differentiated colon associated to cancer DNA hypermethylation. Epigenetics 2009;4:107–13. Richly H, Aloia L, Di Croce L. Roles of the Polycomb group proteins in stem cells and cancer. Cell Death & Disease 2011;2:e204. Riggs MG, Whittaker RG, Neumann JR, Ingram VM. n-Butyrate causes histone modification in HeLa and Friend erythroleukaemia cells. Nature 1977;268:462–4. Ropero S, Fraga MF, Ballestar E, Hamelin R, Yamamoto H, Boix-Chornet M, et al. A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition. Nature Genetics 2006;38:566–9. Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nature Genetics 2000;25: 269–77. Saif MW, Chu E. Biology of colorectal cancer. Cancer Journal 2010;16:196–201. Sakuraba K, Yasuda T, Sakata M, Kitamura YH, Shirahata A, Goto T, et al. Downregulation of Tip60 gene as a potential marker for the malignancy of colorectal cancer. Anticancer Research 2009;29:3953–5. Sakurada K, Furukawa T, Kato Y, Kayama T, Huang S, Horii A. RIZ, the retinoblastoma protein interacting zinc finger gene, is mutated in genetically unstable cancers of the pancreas, stomach, and colorectum. Genes, Chromosomes and Cancer 2001;30:207–11. Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP. A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language. Cell 2011;146:353–8. Santos-Rosa H, Caldas C. Chromatin modifier enzymes, the histone code and cancer. European Journal of Cancer 2005;41:2381–402. Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J, et al. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nature Genetics 2007;39:232–6.

A.N. Gargalionis et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1276–1289 Schnekenburger M, Diederich M. Epigenetics offer new horizons for colorectal cancer prevention. Current Colorectal Cancer Reports 2012;8:66–81. Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, Reuter G, et al. A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes and Development 2004;18:1251–62. Schwartz B, Avivi-Green C, Polak-Charcon S. Sodium butyrate induces retinoblastoma protein dephosphorylation, p16 expression and growth arrest of colon cancer cells. Molecular and Cellular Biochemistry 1998;188:21–30. Siegel R, Naishadham D, Jemal A. Cancer statistics. CA: A Cancer Journal for Clinicians 2012;62:10–29. Sierra J, Yoshida T, Joazeiro CA, Jones KA. The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes and Development 2006;20:586–600. Silva FP, Hamamoto R, Kunizaki M, Tsuge M, Nakamura Y, Furukawa Y. Enhanced methyltransferase activity of SMYD3 by the cleavage of its N-terminal region in human cancer cells. Oncogene 2008;27:2686–92. Sinkkonen L, Hugenschmidt T, Berninger P, Gaidatzis D, Mohn F, Artus-Revel CG, et al. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nature Structural & Molecular Biology 2008;3:259–67. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006;314:268–74. Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer. Nature Reviews Cancer 2006;11:846–56. Spurling CC, Godman CA, Noonan EJ, Rasmussen TP, Rosenberg DW, Giardina C. HDAC3 overexpression and colon cancer cell proliferation and differentiation. Molecular Carcinogenesis 2008;47:137–47. Stark GR, Wang Y, Lu T. Lysine methylation of promoter-bound transcription factors and relevance to cancer. Cell Research 2011;3:375–80. Steele-Perkins G, Fang W, Yang XH, Van Gele M, Carling T, Gu J, et al. Tumor formation and inactivation of RIZ1, an Rb-binding member of a nuclear proteinmethyltransferase superfamily. Genes and Development 2001;15:2250–62. Sun WJ, Zhou X, Zheng JH, Lu MD, Nie JY, Yang XJ, et al. Histone acetyltransferases and deacetylases: molecular and clinical implications to gastrointestinal carcinogenesis. Acta Biochimica et Biophysica Sinica (Shanghai) 2012;44:80–91. Sun Y, Qiao L, Zou B, Xia HH, Gu Q, Ma J, et al. Interactions between XIAP associated factor 1 and a nuclear co-activator, CBP, in colon cancer cells. Digestion 2008;77:79–86. Suzuki K, Suzuki I, Leodolter A, Alonso S, Horiuchi S, Yamashita K, et al. Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell 2006;9:199–207. Tachibana M, Matsumura Y, Fukuda M, Kimura H, Shinkai Y. G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO Journal 2008;27:2681–90. Takawa M, Masuda K, Kunizaki M, Daigo Y, Takagi K, Iwai Y, et al. Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker. Cancer Science 2011;102:1298–305. Tamagawa H, Oshima T, Shiozawa M, Morinaga S, Nakamura Y, Yoshihara M, et al. The global histone modification pattern correlates with overall survival. Oncology Reports 2012;27:637–42. Tanaka H, Hoshikawa Y, Oh-hara T, Koike S, Naito M, Noda T, et al. PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation. Molecular Cancer Research 2009;7:557–69. Taubert S, Gorrini C, Frank SR, Parisi T, Fuchs M, Chan HM, et al. E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1. Molecular and Cellular Biology 2004;24:4546–56. Tay Y, Kats L, Salmena L, Weiss D, Tan SM, Ala U, et al. Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs. Cell 2011;147:344–57. Ting AH, Suzuki H, Cope L, Schuebel KE, Lee BH, Toyota M, et al. A requirement for DICER to maintain full promoter CpG island hypermethylation in human cancer cells. Cancer Research 2008;68:2570–5. Tsuge M, Hamamoto R, Silva FP, Ohnishi Y, Chayama K, Kamatani N, et al. A variable number of tandem repeats polymorphism in an E2F-1 binding element in the 5 flanking region of SMYD3 is a risk factor for human cancers. Nature Genetics 2005;37:1104–7. Tumber A, Collins LS, Petersen KD, Thougaard A, Christiansen SJ, Dejligbjerg M, et al. The histone deacetylase inhibitor PXD101 synergises with 5-fluorouracil

1289

to inhibit colon cancer cell growth in vitro and in vivo. Cancer Chemotherapy and Pharmacology 2007;60:275–83. Uemura M, Yamamoto H, Takemasa I, Mimori K, Hemmi H, Mizushima T, et al. Jumonji domain containing 1A is a novel prognostic marker for colorectal cancer: in vivo identification from hypoxic tumor cells. Clinical Cancer Research 2010;16:4636–46. Vakoc CR, Wen YY, Gibbs RA, Johnstone CN, Rustgi AK, Blobel GA. Low frequency of MLL3 mutations in colorectal carcinoma. Cancer Genetics and Cytogenetics 2009;189:140–1. van Engeland M, Derks S, Smits KM, Meijer GA, Herman JG. Colorectal cancer epigenetics: complex simplicity. Journal of Clinical Oncology 2011;29:1382–91. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 2002;419:624–9. Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, et al. The Polycomb group protein EZH2 directly controls DNA methylation. Nature 2006;439:871–4. Vlaicu SI, Tegla CA, Cudrici CD, Fosbrink M, Nguyen V, Azimzadeh P, et al. Epigenetic modifications induced by RGC-32 in colon cancer. Experimental and Molecular Pathology 2010;88:67–76. Wang CG, Ye YJ, Yuan J, Liu FF, Zhang H, Wang S. EZH2 and STAT6 expression profiles are correlated with colorectal cancer stage and prognosis. World Journal of Gastroenterology 2010;16:2421–7. Wang GG, Allis CD, Chi P. Chromatin remodeling and cancer, part I: covalent histone modifications. Trends in Molecular Medicine 2007;13:363–72. Watanabe T, Kobunai T, Yamamoto Y, Matsuda K, Ishihara S, Nozawa K, et al. Differential gene expression signatures between colorectal cancers with and without KRAS mutations: crosstalk between the KRAS pathway and other signalling pathways. European Journal of Cancer 2011a;47:1946–54. Watanabe Y, Castoro RJ, Kim HS, North B, Oikawa R, Hiraishi T, et al. Frequent alteration of MLL3 frameshift mutations in microsatellite deficient colorectal cancer. PLoS One 2011b;6:e23320. Weichert W, Roske A, Niesporek S, Noske A, Buckendahl AC, Dietel M, et al. Class I histone deacetylase expression has independent prognostic impact in human colorectal cancer: specific role of class I histone deacetylases in vitro and in vivo. Clinical Cancer Research 2008;14:1669–77. Wilson AJ, Velcich A, Arango D, Kurland AR, Shenoy SM, Pezo RC, et al. Novel detection and differential utilization of a c-myc transcriptional block in colon cancer chemoprevention. Cancer Research 2002;62:6006–10. Wilson AJ, Byun DS, Popova N, Murray LB, L‘Italien K, Sowa Y, et al. Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer. Journal of Biological Chemistry 2006;281:13548–58. Wilson AJ, Byun DS, Nasser S, Murray LB, Ayyanar K, Arango D, et al. HDAC4 promotes growth of colon cancer cells via repression of p21. Molecular Biology of the Cell 2008;19:4062–75. Wong CS, Sengupta S, Tjandra JJ, Gibson PR. The influence of specific luminal factors on the colonic epithelium: high-dose butyrate and physical changes suppress early carcinogenic events in rats. Diseases of the Colon and Rectum 2005;48:549–59. Xi Y, Formentini A, Nakajima G, Kornmann M, Ju J. Validation of biomarkers associated with 5-fluorouracil and thymidylate synthase in colorectal cancer. Oncology Reports 2008;19:257–62. Yang XJ. The diverse superfamily of lysine acetyltransferases and their roles in leukemia and other diseases. Nucleic Acids Research 2004;32:959–76. Yuan ZL, Guan YJ, Chatterjee D, Chin YE. Stat3 dimerization regulated by reversible acetylation of a single lysine residue. Science 2005;307:269–73. Zhao Q, Rank G, Tan YT, Li H, Moritz RL, Simpson RJ, et al. PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing. Nature Structural & Molecular Biology 2009;16:304–11. Zhu P, Martin E, Mengwasser J, Schlag P, Janssen KP, Gottlicher M. Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis. Cancer Cell 2004;5:455–63. Zimmermann S, Kiefer F, Prudenziati M, Spiller C, Hansen J, Floss T, et al. Reduced body size and decreased intestinal tumor rates in HDAC2-mutant mice. Cancer Research 2007;67:9047–54. Zuo X, Morris JS, Shureiqi I. Chromatin modification requirements for 15lipoxygenase-1 transcriptional reactivation in colon cancer cells. Journal of Biological Chemistry 2008;283:31341–7.