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Current Opinion in Molecular Therapeutics 2008 10(6): © Thomson Reuters (Scientific) Ltd ISSN 1464-8431
Epigenetic regulation of human epithelial cell cancers
Bradley P Shelton, Neil L Misso, Odette M Shaw, Estri Arthaningtyas & Kanti D Bhoola* Address Lung Institute of Western Australia, Centre for Asthma, Allergy and Respiratory Research, The University of Western Australia, Ground Floor E Block, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, WA 6009, Australia Email:
[email protected] *To whom correspondence should be addressed
Cancer is the second leading cause of death in industrialized countries, with epithelial cell cancers (carcinomas) representing approximately 85% of all diagnosed cancers. The 5-year survival rate for many carcinomas remains low, highlighting the requirement for improved diagnosis and more effective therapies. Epigenetic modifications that do not involve changes in the DNA sequence, but result in changes in gene expression, are rapidly being realized as important in carcinogenesis. Evidence is emerging that DNA methylation, histone modification and alternative mRNA splicing are involved in various human epithelial cell cancers, and diagnostic and therapeutic strategies based on these epigenetic phenomena are under investigation. This review provides an overview of studies demonstrating the importance of epigenetic regulation of gene expression in the diagnosis, progression and response to treatment of human carcinomas. The use of therapeutic agents to reverse these epigenetic changes, either as single treatments or in combination with other therapies, is also discussed. Keywords Alternative splicing, carcinoma, DNA methylation, epigenetic, histone modification, therapy
Introduction Carcinomas, which are cancers derived from epithelial cells, are the most commonly diagnosed form of cancer worldwide [1]. It is well established that carcinomas are associated with genetic changes and dysregulation of gene expression, contributing to both disease development and progression. It has also become apparent that in addition to the well known association between mutations in the DNA sequence and certain carcinomas, epigenetic modifications, that is, changes in gene expression that are not caused by changes in the genetic sequence, also show strong associations with various types of carcinoma. There has consequently been a plethora of research investigating the mechanisms by which epigenetic changes might contribute to altered gene expression, cell cycling and carcinogenesis. Unlike gene mutations, the effects of epigenetic modifications are both developmentally regulated and tissue specific [2], and may be more amenable to reversal in individual patients. Epigenetic changes are therefore regarded as an attractive target for the development of novel, efficacious therapeutic agents for the treatment of cancer. Interest in epigenetics has focused almost exclusively on aberrations of DNA methylation and histone modifications, and advances have already been made in the development of new therapies aimed at reversing the effects of these phenomena. However, a broader view of epigenetics relates to any heritable change in gene expression that does not involve changes in DNA sequence. As such, in reviewing the role of epigenetics in the development of human carcinomas,
it is also necessary to consider the contribution of other epigenetic phenomena; of particular importance is alternative pre-mRNA splicing which, although less commonly studied than DNA methylation and histone modification, may also have a role in carcinogenesis. Similar to DNA methylation and histone modification, advances have been made in establishing methods to correct aberrant alternative splicing associated with tumor development [3]. The development of new therapies to reverse the effects of these epigenetic phenomena is particularly relevant to many types of carcinoma, in which traditional therapeutic approaches are associated with low 5-year survival rates [1]. This review presents an overview of the mechanisms underlying commonly occurring epigenetic changes and their associations with epithelial cancers. In addition, studies demonstrating the potential usefulness of epigenetic modifications as diagnostic and prognostic markers for carcinomas, as well as studies exploring the therapeutic potential of epigenetic regulation of gene expression, are reviewed.
DNA methylation DNA methylation involves the enzyme-mediated covalent addition of a methyl group to cytosine bases that form part of a cytosine-guanine (CpG) dinucleotide. Typically CpG motifs targeted for methylation are located within regions of high CpG content, referred to as CpG islands, which occur in or near the promoter regions of over 70% of all human genes [4]. In normal cells, CpG islands are generally unmethylated, although DNA methylation is involved in normal gene regulatory mechanisms, such as
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genomic imprinting and X chromosome inactivation [5]. In several disease states, most notably cancer, specific CpG islands undergo hypermethylation leading to silencing of the associated genes that are typically involved in suppression of cell growth and proliferation. In many cases both primary tumors and immortalized cancer cell lines have almost identical patterns of hypo- or hypermethylation and these aberrant methylation patterns are distinct from those of healthy tissue [6,7]. The epigenetic mechanisms associated with cancer and their clinical significance have been reviewed in references [2,8].
Hypomethylation A characteristic feature of most human cancers is global hypomethylation of the genome [9,10], although this may vary between individuals from different ethnic backgrounds [11]. It is likely that global hypomethylation plays a role in the development and progression of cancer by increasing genomic instability and the frequency of chromosomal rearrangements [12]. In 2006, Suzuki et al demonstrated an age-dependent accumulation of DNA demethylation in gastrointestinal cancer that preceded the onset of genomic damage, suggesting that DNA hypomethylation is an early event in the development of carcinogenesis [13]. Genome-wide hypomethylation, together with CpG hypermethylation, has been correlated with both biological features and clinical outcomes of hepatocellular carcinoma, suggesting hypomethylation is a prognostic marker for tumor development [14]. Similar results have been observed in squamous cell carcinomas of the head and neck [15] and breast carcinoma [16]. In addition, global hypomethylation in leukocytes has been identified as a risk factor for the development of bladder cancer, independent of smoking history [17]. In addition to generalized hypomethylation, gene-specific hypomethylation may also lead to altered gene expression and potential deregulation of cell growth. Overexpression of the synuclein γ gene, also known as breast cancerspecific gene 1, has previously been shown to contribute to breast and ovary carcinogenesis. In 2003, Gupta et al identified hypomethylation of exon 1 of the synuclein γ gene as a potential mechanism leading to the overexpression of this gene in both breast and ovarian cancer cell lines [18]. In addition, hypomethylation of the protein gene product 9.5 (PGP9.5) promoter contributed to increased expression of this gene in gall bladder cancer [19]. As PGP9.5 was previously suggested to be a potential marker of non-small-cell lung carcinoma [20] and esophageal squamous cell carcinoma [21], it is possible that hypomethylation of this gene may also be associated with these carcinomas. Hypomethylation may also regulate expression of the cyclin D2 gene, which was overexpressed in a subset of gastric carcinomas [22]; cyclin D2 hypomethylation was identified in both gastric carcinoma cell lines and tumor samples [23]. Furthermore, treatment of cyclin D2-negative cells with the demethylating agent 5-aza-2'-deoxycytidine increased cyclin D2 expression, suggesting demethylation as a
potential mechanism for mediating the upregulation of this gene in cancer [23]. DNA hypomethylation may also regulate expression of chemoresistance-related genes, including multidrug resistance 1 (MDR1), glutathione-S-transferase pi (GSTP1) and O6-methylguanine DNA methyltransferase (MGMT). The expression of each of these genes is downregulated by promoter hypermethylation in several human carcinomas and carcinoma cell lines, including the breast adenocarcinoma cell line, MCF-7 [24-27]. In 2006, Chekhun et al compared the methylation status of MDR1, GSTP1 and MGMT in MCF-7 cells with a doxorubicinresistant MCF-7 variant [28]. This study identified a significant decrease in the methylation status of each gene in the drug-resistant cell line, suggesting that hypomethylation of these genes may contribute to the development of doxorubicin resistance. These data suggest that hypomethylation may contribute to the development of drug resistance in other carcinomas.
Hypermethylation Most human carcinomas display some level of DNA hypermethylation, with an associated transcriptional silencing of gene expression [29,30]. Genes silenced by hypermethylation in cancer are involved in a variety of cellular processes, such as cell-cycle regulation, transcriptional regulation, DNA repair, apoptosis, Ras signaling, cell adhesion and invasion, and response to carcinogens [31]. While the pathways affected by DNA hypermethylation are common to various types of cancer, the silencing of specific genes varies considerably between different carcinomas, as well as between different subtypes and stages of the same carcinoma [32]. Because of the rapid explosion of studies on gene regulation by DNA methylation, it is beyond the scope of this review to summarize all data that may be pertinent to epithelial cell carcinomas. Instead, an overview is provided of some of the more significant and interesting studies.
Cell cycle progression Hypermethylation of p16, which codes for a protein inhibitor of cyclin D1 that is involved in regulating cell cycle progression, has been demonstrated in colorectal cancer [33], gastric cancer [34], lung cancer [26,35] esophageal squamous cell carcinoma [36] and hepatocellular carcinomas [37]. The relative frequency of p16 silencing by methylation suggests that this is a true oncogenic event, rather than the consequence of carcinogenesis. Interestingly, several studies have demonstrated that methylation of p16 was associated with exposure to environmental carcinogens and Georgiou et al showed increased methylation of p16 in lung epithelium of heavy smokers compared with non-smokers [38]. Methylation of p16 in circulating DNA may also be a diagnostic marker for some carcinomas [39]. While the role of p16 methylation in the development of cancer is an area of great interest (eg, p14, p15 and p27 are also hypermethylated in some forms of cancer), the silencing of these genes is not as frequent as that of p16 [40,41].
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Transcriptional regulation Another family of genes that are differentially methylated in various carcinomas are members of the GATA family of transcription factors. The expression of GATA6 is decreased by hypermethylation in ovarian cancer cell lines [42], but expression is normal in esophageal and lung cancer cell lines [43,44]. In contrast, studies have shown that GATA4 and GATA5 were methylated in 67 and 41%, respectively, of primary lung tumors [44], 61 and 32% of esophageal squamous carcinomas and 71 and 55% of esophageal adenocarcinomas [43]. SOX2, another transcription factor, is usually expressed in gastric epithelial cells, but was silenced by promoter hypermethylation in gastric cancer cell lines and in almost 20% of primary gastric tumors [45]. The same study also showed that hypermethylation of SOX2 was associated with significantly decreased survival in patients with advanced gastric cancer [45].
DNA repair Epigenetic silencing of MGMT, a DNA repair enzyme, by DNA methylation was observed in lung and esophageal primary tumors, and in 50% of lung cancer cell lines investigated [24,26]. MGMT removes alkyl groups from O6-methylguanine, preventing mismatch with thymine and potential DNA damage. Inactivation of MGMT is thought to allow the accumulation of mismatch mutations in both oncogenes and tumor suppressor genes, contributing to the process of carcinogenesis. Therefore downregulation of MGMT may occur at an early stage in cancer development. In support of this, methylation of MGMT was detected in over 50% of premalignant lesions of the colorectal mucosa [46]. If epigenetic silencing of MGMT is an early event in carcinogenesis, the methylation status of the MGMT promoter may prove to be a useful marker for the early detection of carcinoma. The nucleotide excision repair gene, xeroderma pigmentosum group C (XPC), has demonstrated hypermethylation in lung cancer cell lines and in one third of clinical lung tumor samples [47]. Interestingly, inactivation of XPC by hypermethylation was more common in patients with lung cancer with no smoking history. In vitro studies demonstrated that inactivation of XPC led to mutations in the p53 gene [47], suggesting that epigenetic silencing of XPC may be an early event in carcinogenesis. However, to date, methylation of XPC has not been observed in other carcinomas.
Apoptosis Gopisetty et al suggested that many of the genes silenced by hypermethylation are associated with the apoptosis pathway [48]. An important apoptosis-related gene regulated by DNA methylation is death-associated protein kinase (DAPK). DAPK was methylated in 77% of transitional cell carcinomas of the bladder and 33% of renal cell carcinomas [49], as well as in 60% of Barrett's esophageal adenocarcinomas [50]. In clear cell renal carcinoma, an association between DAPK methylation and more aggressive disease was demonstrated [51]. DAPK was also methylated in patients with NSCLC, although there was
no association with smoking status, tumor histology or tumor stage [52]. In contrast, methylation of DAPK in cervical cancer was associated with both the histological type of squamous cell carcinoma and advanced tumor stage [53]. In addition, methylation of DAPK may predict prognosis and response to chemotherapy in patients with gastric cancer [54].
Ras signaling The Ras association domain family 1A (RASSF1A) gene, which is thought to encode a Ras effector protein, is downregulated by hypermethylation in cervical [55], prostate [56], breast [57] and small cell cancers, as well as NSCLC [58,59]. Methylation of RASSF1A has been detected in circulating DNA in the serum of 34% of patients with lung cancer [58] and 75% of patients with breast cancer [57]. A dose-dependent increase in RASSF1A methylation was observed in mice exposed to arsenic, suggesting that methylation of RASSF1A may be a consequence of exposure to carcinogens [60]. Dammann et al demonstrated that methylation of RASSF1A in lung cancer was associated with exposure to cigarette smoke [61], although other data have not revealed an association with smoking status [52].
Cell adherence and invasion Inactivation of cadherin-mediated cell adhesion is a feature of several cancers. The gene encoding E-cadherin (CDH1), is silenced by promoter hypermethylation in gastric [62] and bladder cancers [63]. Significantly, CDH1 hypermethylation is also associated with infiltrating breast cancer, suggesting a role for CDH1 methylation in metastasis [64]. Simultaneous methylation of CDH1 and the H-cadherin gene, CDH13, has been suggested as a potential prognostic marker in patients with NSCLC [65]. Tissue inhibitor of metalloprotease-3 (TIMP-3) regulates matrix metalloprotease activity and is likely important in cancer cell invasion [66]. The TIMP-3 gene is downregulated by DNA hypermethylation in several cancers [67,68] and the consequent dysregulation of matrix metalloprotease activity may increase tumor invasiveness. Hypermethylation of TIMP-3 is associated with invasive breast ductal carcinoma [69] and colorectal carcinoma [70], and decreased TIMP-3 methylation has also been identified as an independent prognostic factor for survival among patients with bladder cancer [71]. A recent 3-dimensional (3-D) microarray study identified methylation of TIMP-3 in 43%, p16 in 38%, CDH13 in 54%, DAPK in 50%, CDH1 in 11%, RASSF1 in 18% and the gene encoding the CD44 cell adhesion molecule in 18% of primary NSCLC samples (n = 28), whereas methylation was not observed in corresponding non-malignant tissues [72]. This study demonstrated that 3-D microarray provided a potentially useful HTS platform for analysis of DNA hypermethylation in tumor tissue.
Detoxification The GSTP1 gene encodes the glutathione S-transferase pi dei was the most commonly identified genetic alteration
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in prostate cancer, with > 90% of these carcinomas demonstrating decreased GSTP1 expression (reviewed in reference [73]). As GSTP1 is also hypermethylated in pre-cancerous lesions of the prostate, silencing of this gene may be an early event in carcinogenesis, making it a potential target for chemopreventative treatment. Downregulation of GSTP1 expression by hypermethylation has also been observed as an early event in breast carcinogenesis [27] and the gene was methylated in 15% of NSCLC in a Korean population [74].
Clinical and therapeutic potential The identification of specific methylation events occurring early in carcinogenesis and the suggestion that epigenetic silencing of tumor suppressor genes plays a causative role in cancer development, have generated significant interest in the use of DNA methylation as a diagnostic marker of disease. This is particularly relevant in view of the identification of cancer-specific gene methylation in DNA from serum and sputum samples, increasing the potential for non-invasive diagnosis. Promoter methylation in sputum and serum samples was investigated as a potential diagnostic marker for NSCLC, and revealed a strong correlation between methylation of sputum DNA and methylation of the same genes in the tumor [75]. However, promoter methylation in sputum samples was also associated with a high false-positive rate, demonstrating the requirement for additional research before methylation-based diagnostic tools can be utilized in a clinical setting. Potential epigenetic markers for specific cancers are summarized in reference [2]. Analysis of promoter methylation may also provide useful prognostic markers of disease progression and response to therapy. Specific methylation events have been associated with different tumor stages and with poorer prognoses [76]. Epigenetic changes are also under investigation as predictors of response to traditional chemotherapy. For example, methylation of p16 predicted improved responses to 5-fluorouracil-based adjuvant chemotherapy in patients with gastric cancer [77]. Conversely, epigenetic modification of the multidrug resistance MDR1 gene by chemotherapeutic agents may lead to the development of multidrug resistance in cancer [78]. The reversibility of DNA methylation has led to the development of novel therapies based on restoring the activity of silenced genes by inhibiting DNA methylation. Currently, the most widely investigated inhibitors of methylation are the nucleoside analogs, 5-azacytidine, 5-aza-2'-deoxycytidine and the chemically stable cytidine analog, 1-(β-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine; University of Oregon/University of Southern California), which are incorporated into DNA, preventing the activity of DNA methyltransferases (DNMT) [79]. Although these agents cause global hypomethylation, they typically only partially reactivate the transcription of silenced genes [80]. However, the global hypomethylation produced by nucleoside inhibitors of methylation is also
a hallmark of most cancers. Therefore, reduced activity of DNA methyltransferases may be associated with the promotion of tumor development due to the disruption of essential methylation and chromosomal instability [81]. Although nucleoside inhibitors have progressed to clinical trials in several cancers [82], treatment outcomes appear to have been improved in hematopoietic cancers than for solid tumors [83]. The toxicity of nucleoside analogs has led to the investigation of other specific inhibitors of DNMT activity, including procainamide and procaine (Pharmaplaz Ltd); hydralazine: the green tea polyphenol, epigallocatechin3-gallate (Anagen Therapeutics Inc); the novel small molecule RG-108; and antisense oligodeoxynucleotides [84]. One antisense oligodeoxynucleotide, MG-98 (MGI Pharma Inc), targets DNMT1, and has shown promising results in phase I clinical trials, although these have not been translated into success in phase II clinical trials in advanced metastatic renal carcinoma [85].
Chromatin remodeling In addition to DNA methylation, transcriptional regulation of genes is also affected by changes in DNA packaging. Within the chromosome, chromatin fibers are wrapped around a complex of histone proteins, forming the nucleosome. Each histone core consists of two subunits each of the proteins H2A, H2B, H3 and H4, forming an octamer of these basic proteins. DNA is usually tightly bound to the histone core, preventing transcription of the encoded genes. However, this transcriptional regulation is reversible through chromatin remodeling, which occurs by several mechanisms, including structural alteration of chromatin, histone replacement and histone modifications, such as phosphorylation, methylation, and acetylation; chromatin remodeling therefore regulates gene expression by altering the interaction between DNA and the histone proteins [86]. One of the more extensively studied histone modifications is acetylation of lysine residues at the N-terminal ends of histone proteins [87]. Addition of these acetyl groups results in the loss of positive charge, disrupting the interaction between chromatin and DNA and leading to transcriptional activation. Lysine residues may also be methylated, with the effect on transcription depending on the target lysine [88]. Similar to DNA methylation, dysregulation of chromatin remodeling is associated with carcinogenesis.
Histone deacetylation Histone deacetylases (HDACs) regulate the expression and activity of various proteins involved in both cancer initiation and cancer progression. By removal of acetyl groups from lysine residues within histones, HDACs create a non-permissive chromatin conformation that prevents the transcription of genes encoding proteins involved in tumorigenesis. Increased activity of HDAC has been observed in a range of human carcinomas, including breast, gastric, esophageal and colon cancers [89]. In addition to histones, HDACs bind to and deacetylate a variety of other protein targets likely to be involved in
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critical regulatory processes, including cell growth, differentiation and survival, transcription factors, apoptosis, angiogenesis and immunogenicity [90]. Downregulation of the GATA4 and GATA6 transcription factors in ovarian cancer cell lines has been associated with hypoacetylation of histones H3 and H4, and expression of these proteins was restored by treatment with the HDAC inhibitor, trichostatin A [42].
Histone methylation The role of histone methylation in regulating gene expression is more complex than that of histone deacetylation. Histone methylation, mediated by histone methyl transferases, may either activate or repress gene transcription, depending on which lysine residue is methylated. Methylation of lysines 9, 27 and 36 within H3 and lysine 20 of H4 is associated with transcriptional silencing, while methylation of lysines 4 and 79 of H3 is associated with transcriptional activation [88]. As an additional level of complexity, histone methylation may occur as mono-, di- or trimethylation [91]. Although not as widely associated with carcinogenesis as histone acetylation, histone methylation has been identified as playing a role in the progression of some cancers; for example, the loss of trimethylation of lysine 20 in H4 was observed early in oncogenic transformation and increased with tumor progression [92].
Therapeutic potential The potential of histone deacetylase inhibitors as therapeutic agents has received considerable attention. The mechanisms of action and clinical significance of this class of compounds have been reviewed in references [93,94]. Considerable attention has been focused on the use of HDAC inhibitors, such as hydroxamic acids, aliphatic acids, benzamides and cyclic peptides, as anticancer therapies. Hydroxamic acids, such as trichostatin A and vorinostat (suberoylanilide hydroxamic acid), non-selectively inhibit both class I and class II HDACs. While trichostatin A is commonly utilized in research, its use as a cancer therapy is limited by its toxicity and low bioavailability [95]. This has led to the development of synthetic agents based on the structure of trichostatin A [95]. The hydroxamic acids are generally efficacious at low micromolar to nanomolar concentrations and, in the case of vorinostat, can be delivered orally [90]. Vorinostat has received FDA approval for the treatment of patients with cutaneous T-cell lymphoma [89], and is currently in several phase II clinical trials for the treatment of carcinomas, both as a single agent and in combination with other anticancer therapies [96]. Results from clinical trials in ovarian, peritoneal, head and neck, breast, colorectal and lung carcinomas demonstrated that vorinostat was well tolerated as a single therapy, but had minimal anticancer activity [97-99]. The limited efficacy of vorinostat may be due to upregulation of the anti-apoptotic transcription factor NFκB [100]. In support of this, the use of vorinostat in combination with gemcitabine decreased NFκB
activation and increased apoptosis of NSCLC cell lines, suggesting improved therapeutic potential for vorinostat if used in combination with conventional chemotherapeutic agents [101]. Cyclic peptides, including apicidin and romidepsin (depsipeptide/FK-228, Gloucester Pharmaceuticals Inc; Figure 1), are another class of HDAC inhibitors that have received considerable attention as potential anticancer treatments. These agents demonstrated effective doses comparable to those of the hydroxamic acids [95]. Early in vitro and in vivo data for romidepsin suggested that this compound had novel anti-angiogenic activity and could suppress tumor expansion by inhibiting neovascularization [102]. However, a phase II clinical trial of romidepsin in metastatic clear cell renal carcinoma demonstrated objective responses to treatment in only 7% of patients [103], compared with a response rate of 11% among patients with metastatic renal cell cancer enrolled in a phase II clinical trial of gemcitabine plus capecitabine [104]. Romidepsin may be more effective when used in combination with flavopiridol, an inhibitor of the cyclin-dependent kinase, p21, as flavopiridol enhanced romidepsin-mediated apoptosis of lung and esophageal cancer cells [105]. The use of HDAC inhibitors as antitumor therapies may be improved by increasing their specificity, particularly if HDACs crucial for tumorigenesis can be identified and targeted [95]. The development of such therapeutic agents may involve either rational drug design or the use of antisense oligonucleotides, in a manner analogous to the use of antisense oligonucleotides to inhibit DNMT [85].
Alternative splicing Alternative splicing refers to the mechanism by which pre-mRNA molecules transcribed from a single gene are spliced in varying combinations of exons and introns to produce several transcripts that may be translated into distinct proteins [106]. Genome-wide analyses suggest that up to 80% of human genes give rise to alternatively spliced forms [107]. Although most aberrant mRNAs are degraded, some splice variants may be translated into distinct protein isoforms with altered or even antagonistic
Figure 1. The structure of romidepsin. H3C O H N
H3C
NH S S O NH
O
H O
H3C
CH3 O O
H NH CH3
Romidepsin (Gloucester Pharmaceuticals)
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functions [108]. There is increasing evidence that alternative and aberrant pre-mRNA splicing, resulting from the creation or disruption of splice sites or the effects of splicing enhancers and silencers, may have an important role in human cancers [109]. Alternative splicing of key genes may critically impact on major aspects of cancer cell biology, including cell-cell and cell-matrix interactions, metastasis, angiogenesis and apoptosis [110].
Alternative splicing in cancer One of the most intensively studied alternatively spliced genes associated with cancer is CD44, a transmembrane protein involved in cell-cell and cell-matrix interactions. This gene has more than 20 known isoforms that are produced by variable incorporation of ten alternative exons in its extracellular domain [111]; expression of these multiple isoforms may alter processes such as lymphocyte recruitment, epithelial cell-matrix adhesion, and tumor metastasis [112]. Immunohistochemical studies showed that the CD44 isoform containing the variable exon 6 (CD44v6) was often upregulated in squamous cell carcinomas, but was rarely expressed in non-epithelial tumors [113]. This expression pattern suggests that CD44v6 may be a potential target for antibody-based therapy in various types of epithelial cancers [113]. Alternative splicing of the Rho-family GTPase, Rac1, generates Rac1b through inclusion of a 57-nucleotide exon [114]. Rac1b accumulated in colorectal and breast tumors, and demonstrated transforming properties when overexpressed in cultured cells [114]. VEGF-A is an alternatively spliced gene important in tumor angiogenesis and is upregulated in almost all solid tumors [115]. VEGF-A stimulates endothelial cell proliferation and migration by binding to its receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1) [116]. The human VEGF-A gene is spliced into nine angiogenic isoforms, with VEGF121, VEGF165 and VEGF189 being the most abundant transcripts in various tissues [117]. VEGF189 includes exons 6 and 7, which encode a cationic domain that binds to heparin and is responsible for its strong affinity for extracellular matrix (ECM). VEGF165 lacks exon 6, which results in a weaker interaction with ECM and a higher diffusibility than VEGF189 [116]. VEGF121 lacks both exons 6 and 7 and is therefore the most diffusible isoform [116]. The VEGF121 isoform is thought to be important for the recruitment of large extra-tumoral systemic vessels, whereas the heparin-binding isoforms, such as VEGF189, seem to have a role in internal tumor microvascularization [118]. VEGF121 demonstrated a greater role in tumor vascularization in xenograft models of breast cancer than VEGF165 or VEGF189 [119]. Survivin and p53 are examples of the large number of apoptosis-related genes regulated by alternative splicing. p53 is a crucial protein in cell cycle regulation and apoptosis, and multiple isoforms are generated through two different promoters and by alternative splicing [120]. These isoforms are expressed in a tissue-dependent
manner and show altered expression in human breast tumors [120]. Survivin is an anti-apoptotic oncogene that was highly upregulated in various tumors and may be a novel therapeutic target [121]. Alternative splicing of survivin pre-mRNA produces three different mRNAs, which encode distinct proteins that differ in their anti-apoptotic properties [122]. The survivin-2B variant, which has pro-apoptotic properties and acts as a naturally occurring antagonist of survivin, is downregulated in late-stage or metastatic gastric cancer [123]. Expression of survivin-2B is also higher in small breast tumors, while survivin-3B is more frequently expressed in breast carcinomas with a p53 mutation, suggesting that survivin-3B may be an anti-apoptotic factor in breast carcinoma and that its expression is regulated by p53 [124].
Therapeutic potential The discovery of cancer-specific alternatively spliced proteins has created interest in their use as cancer biomarkers, both at the mRNA and protein levels [125]. Associations between differential expression of splicing isoforms and tumor progression have been demonstrated for proteins such as survivin [122]. VEGF protein isoforms extracted from tumor lysates were prognostic for survival in patients with node-positive breast cancer [126]. However, additional studies are required to demonstrate the independent prognostic value of splice variants in cancer. Progress in biomarker research is expected to be advanced by HTS technologies, such as splicing-specific microarrays, that help to define the expression profiles of multiple genes associated with disease progression or response to treatment. For example, a splicing array was utilized to examine more than 1500 mRNA isoforms from a panel of genes previously associated with prostate cancer [127]. A large number of cell-type-specific mRNA isoforms were identified, and extensive covariation between transcription and splicing in prostate cancer cells was demonstrated [127]. Alternative splicing is also a target for molecular therapies. Therapeutic agents could be specifically designed to target splice variants that enhance disease processes or to correct malfunctioning splice regulatory mechanisms. Antisense RNA, siRNA, and ribozymes are exogenous oligonucleotides that can be designed to recognize aberrant mRNA molecules and rectify their splicing [128]. Treatments based on such oligonucleotides may in future be utilized to specifically target mRNA isoforms of genes that are important in cancer development and progression. Synthetically modified oligonucleotides (SMO) repress gene translation and block the spliceosome machinery at specific sites [129]. A novel antisense oligonucleotide was utilized to inhibit the anti-apoptotic Bcl-2 family genes and induce apoptosis in prostate cancer cells [130]. This Bcl-2/Bcl-xL bispecific antisense oligonucleotide also enhanced the chemosensitivity of the cells to paclitaxel
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[130]. Oblimersen (Genasense; Genta Inc) is an SMO that inhibits Bcl-2 translation, and is currently undergoing phase II and phase III clinical trials for several types of malignancies [131]. In a randomized controlled phase III clinical trial, the addition of oblimersen to dacarbazine significantly improved multiple clinical outcomes in patients with advanced melanoma [132]. Antisense strategies are also under investigation for other targets, such as survivin and clusterin [133]. Although there are still questions relating to the pharmacology of SMO that need to be resolved, the latest generation of antisense oligonucleotides are well tolerated and safe, and have been shown to modulate the expression of target proteins in vivo [134]. Therefore, SMO-based strategies that target specific splicing alterations could in future be utilized for cancer treatment, as has been demonstrated experimentally in other splicing-related pathologies, such as Duchenne's muscular dystrophy [135].
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Conclusions Epigenetic modifications in epithelial cell cancers include DNA methylation, histone acetylation, chromatin remodeling and deregulated signaling pathways, which are potential targets for molecular therapies. Epigenetic modifications that regulate gene expression are increasingly recognized as playing a critical role in the development of human cancers, including carcinomas. Identification of the specific epigenetic changes that precede the development of carcinomas and are associated with disease progression could form the basis for developing potentially useful diagnostic and prognostic disease markers. However, the greatest interest in epigenetic modifications in relation to cancer has been focused on the development of new therapeutic strategies. Anticancer therapies based on the reversal of epigenetic changes have shown significant potential in preclinical trials. Despite this, both demethylating agents and HDAC inhibitors have demonstrated limited success in clinical trials for the treatment of various carcinomas. The limitations of these treatments may be overcome and efficacy improved by targeting treatments to specific DNMTs and HDACs, for example, by the use of antisense oligonucleotides targeting DNMT1. However, the potential adverse consequences of demethylating agents and HDAC inhibitors with respect to global hypomethylation and histone acetylation also need to be addressed. Therapies aimed at correcting aberrant alternative splicing have shown success in modulating target protein expression in patients. However, the highly gene-specific nature of these therapies, together with the genetic complexity of cancer, is likely to limit the usefulness of such therapeutic strategies in the treatment of carcinomas. In this regard, the development of combination therapies based on synthetically modified oligonucleotides that target several genes simultaneously is a potentially promising area of investigation. This review has focused on major advances in molecular studies of the origin and biology of epigenetic changes in epithelial cell cancers.
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10. Tryndyak VP, Kovalchuk O, Pogribny IP: Loss of DNA methylation and histone H4 lysine 20 trimethylation in human breast cancer cells is associated with aberrant expression of DNA methyltransferase 1, Suv4-20h2 histone methyltransferase and methyl-binding proteins. Cancer Biol Ther (2006) 5(1):65-70. 11. Piyathilake CJ, Henao O, Frost AR, Macaluso M, Bell WC, Johanning GL, Heimburger DC, Niveleau A, Grizzle WE: Race- and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung (United States). Cancer Causes Control (2003) 14(1):37-42. 12. Robertson KD: DNA methylation and human disease. Nat Rev Genet (2005) 6(8):597-610. 13. Suzuki K, Suzuki I, Leodolter A, Alonso S, Horiuchi S, Yamashita K, Perucho M: Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell (2006) 9(3):199-207. 14. Calvisi DF, Ladu S, Gorden A, Farina M, Lee JS, Conner EA, Schroeder I, Factor VM, Thorgeirsson SS: Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest (2007) 117(9):2713-2722. 15. Smith IM, Mydlarz WK, Mithani SK, Califano JA: DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage. Int J Cancer (2007) 121(8):1724-1728. 16. Soares J, Pinto AE, Cunha CV, André S, Barão I, Sousa JM, Cravo M: Global DNA hypomethylation in breast carcinoma: Correlation with prognostic factors and tumor progression. Cancer (1999) 85(1):112-118. 17. Moore LE, Pfeiffer RM, Poscablo C, Real FX, Kogevinas M, Silverman D, García-Closas R, Chanock S, Tardón A, Serra C, Carrato A et al: Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: A case-control study. Lancet Oncol (2008) 9(4):359-366.
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35. Brock MV, Hooker CM, Ota-Machida E, Han Y, Guo M, Ames S, Glöckner S, Piantadosi S, Gabrielson E, Pridham G, Pelosky K et al: DNA methylation markers and early recurrence in stage I lung cancer. N Engl J Med (2008) 358(11):1118-1128. • An interesting study showing that promoter methylation of four genes, including p16, was associated with early recurrence of lung cancer. 36. Fujiwara S, Noguchi T, Takeno S, Kimura Y, Fumoto S, Kawahara K: Hypermethylation of p16 gene promoter correlates with loss of p16 expression that results in poorer prognosis in esophageal squamous cell carcinomas. Dis Esophagus (2008) 21(2):125-131. 37. Nishida N, Nagasaka T, Nishimura T, Ikai I, Boland CR, Goel A: Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology (2008) 47(3):908-918. 38. Georgiou E, Valeri R, Tzimagiorgis G, Anzel J, Krikelis D, Tsilikas C, Sarikos G, Destouni C, Dimitriadou A, Kouidou S: Aberrant p16 promoter methylation among Greek lung cancer patients and smokers: Correlation with smoking. Eur J Cancer Prev (2007) 16(5):396-402. 39. Wong TS, Man MW, Lam AK, Wei WI, Kwong YL, Yuen AP: The study of p16 and p15 gene methylation in head and neck squamous cell carcinoma and their quantitative evaluation in plasma by real-time PCR. Eur J Cancer (2003) 39(13):1881-1887. 40. Esteller M, Herman JG: Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol (2002) 196(1):1-7. 41. Auerkari EI: Methylation of tumor suppressor genes p16INK4a, p27Kip1 and E-cadherin in carcinogenesis. Oral Oncol (2006) 42(1):5-13. 42. Caslini C, Capo-chichi CD, Roland IH, Nicolas E, Yeung AT, Xu XX: Histone modifications silence the GATA transcription factor genes in ovarian cancer. Oncogene (2006) 25(39):5446-5461. 43. Guo M, House MG, Akiyama Y, Qi Y, Capagna D, Harmon J, Baylin SB, Brock MV, Herman JG: Hypermethylation of the GATA gene family in esophageal cancer. Int J Cancer (2006) 119(9):2078-2083. 44. Guo M, Akiyama Y, House MG, Hooker CM, Heath E, Gabrielson E, Yang SC, Han Y, Baylin SB, Herman JG, Brock MV: Hypermethylation of the GATA genes in lung cancer. Clin Cancer Res (2004) 10(23):7917-7924. 45. Otsubo T, Akiyama Y, Yanagihara K, Yuasa Y: SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis. Br J Cancer (2008) 98(4):824-831. • A study in which the potential importance of SOX2 methylation in gastric cancer was demonstrated. 46. Menigatti M, Pedroni M, Verrone AM, Borghi F, Scarselli A, Benatti P, Losi L, Di Gregorio C, Schär P, Marra G, Ponz de Leon M et al: O6-methylguanine-DNA methyltransferase promoter hypermethylation in colorectal carcinogenesis. Oncol Rep (2007) 17(6):1421-1427. 47. Wu YH, Tsai Chang JH, Cheng YW, Wu TC, Chen CY, Lee H: Xeroderma pigmentosum group C gene expression is predominantly regulated by promoter hypermethylation and contributes to p53 mutation in lung cancers. Oncogene (2007) 26(33):4761-4773. 48. Gopisetty G, Ramachandran K, Singal R: DNA methylation and apoptosis. Mol Immunol (2006) 43(11):1729-1740. 49. Christoph F, Kempkensteffen C, Weikert S, Köllermann J, Krause H, Miller K, Schostak M, Schrader M: Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer. Br J Cancer (2006) 95(12):1701-1707. 50. Kuester D, Dar AA, Moskaluk CC, Krueger S, Meyer F, Hartig R, Stolte M, Malfertheiner P, Lippert H, Roessner A, El-Rifai W et al: Early involvement of death-associated protein kinase promoter hypermethylation in the carcinogenesis of Barrett's esophageal adenocarcinoma and its association with clinical progression. Neoplasia (2007) 9(3):236-245.
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51. Christoph F, Hinz S, Kempkensteffen C, Schostak M, Schrader M, Miller K: mRNA expression profiles of methylated APAF-1 and DAPK-1 tumor suppressor genes uncover clear cell renal cell carcinomas with aggressive phenotype. J Urol (2007) 178(6):2655-2659. 52. Liu Y, Gao W, Siegfried JM, Weissfeld JL, Luketich JD, Keohavong P: Promoter methylation of RASSFIA and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and neversmokers. BMC Cancer (2007) 7:74. 53. Leung RC, Liu SS, Chan KY, Tam KF, Chan KL, Wong LC, Ngan HY: Promoter methylation of death-associated protein kinase and its role in irradiation response in cervical cancer. Oncol Rep (2008) 19(5):1339-1345. 54. Kato K, Iida S, Uetake H, Takagi Y, Yamashita T, Inokuchi M, Yamada H, Kojima K, Sugihara K: Methylated TMS1 and DAPK genes predict prognosis and response to chemotherapy in gastric cancer. Int J Cancer (2008) 122(3):603-608. 55. Yu MY, Tong JH, Chan PK, Lee TL, Chan MW, Chan AW, Lo KW, To KF: Hypermethylation of the tumor suppressor gene RASSFIA and frequent concomitant loss of heterozygosity at 3p21 in cervical cancers. Int J Cancer (2003) 105(2):204-209. 56. Yegnasubramanian S, Kowalski J, Gonzalgo ML, Piantadosi S, Walsh PC, Bova GS, De Marzo AM, Nelson WG: Hypermethylation of CpG islands and metastatic human prostate cancer. Cancer 64(6):1975-1986.
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67. Gu P, Xing X, Tänzer M, Röcken C, Weichert W, Ivanauskas A, Pross M, Peitz U, Malfertheiner P, Schmid RM, Ebert MP: Frequent loss of TIMP-3 expression in progression of esophageal and gastric adenocarcinomas. Neoplasia (2008) 10(6):563-572. 68. Feng H, Cheung AN, Xue WC, Wang Y, Wang X, Fu S, Wang Q, Ngan HY, Tsao SW: Down-regulation and promoter methylation of tissue inhibitor of metalloproteinase 3 in choriocarcinoma. Gynecol Oncol (2004) 94(2):375-382. 69. Lui EL, Loo WT, Zhu L, Cheung MN, Chow LW: DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma. Biomed Pharmacother (2005) 59(Suppl 2):S363-S365. 70. Brueckl WM, Grombach J, Wein A, Ruckert S, Porzner M, Dietmaier W, Rümmele P, Croner RS, Boxberger F, Kirchner T, Hohenberger W et al: Alterations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) are found frequently in human colorectal tumours displaying either microsatellite stability (MSS) or instability (MSI). Cancer Lett (2005) 223(1):137-142. 71. Hoque MO, Begum S, Brait M, Jeronimo C, Zahurak M, Ostrow KL, Rosenbaum E, Trock B, Westra WH, Schoenberg M, Goodman SN et al: Tissue inhibitor of metalloproteinases-3 promoter methylation is an independent prognostic factor for bladder cancer. J Urol (2008) 179(2):743-747. 72. Wang Y, Zhang D, Zheng W, Luo J, Bai Y, Lu Z: Multiple gene methylation of non small cell lung cancers evaluated with 3-dimensional microarray. Cancer (2008) 112(6):1325-1336. • An interesting study in which a 3-D microarray technique was utilized to assess the methylation status of multiple genes in tumor tissue. 73. Meiers I, Shanks JH, Bostwick DG: Glutathione S-transferase pi (GSTP1) hypermethylation in prostate cancer: Review 2007. Pathology (2007) 39(3):299-304. •• A useful overview of the significance of GSTP1 methylation for the diagnosis and possible prevention of prostate cancer. 74. Kim DS, Cha SI, Lee JH, Lee YM, Choi JE, Kim MJ, Lim JS, Lee EB, Kim CH, Park TI, Jung TH et al: Aberrant DNA methylation profiles of non-small cell lung cancers in a Korean population. Lung Cancer (2007) 58(1):1-6. 75. Belinsky SA, Grimes MJ, Casas E, Stidley CA, Franklin WA, Bocklage TJ, Johnson DH, Schiller JH: Predicting gene promoter methylation in non-small-cell lung cancer by evaluating sputum and serum. Br J Cancer (2007) 96(8):1278-1283. • This study demonstrated that sputum analysis provided a non-invasive strategy to predict gene promoter methylation status in lung cancer. 76. Esteller M: Epigenetics 358(11):1148-1159.
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