Contemporary proteomic strategies for clinical epigenetic research and potential impact for the clinic.

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Contemporary proteomic strategies for clinical epigenetic research and potential impact for the clinic Expert Rev. Proteomics 12(2), 197–212 (2015)

Petra Hudler*, Alja Videticˇ Paska and Radovan Komel* Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia *Authors for correspondence: Tel.: +386 1 543 76 63 Fax: +386 1 543 76 41 [email protected]; [email protected]

Novel proteomic methods are revealing the intricacy of the epigenetic landscape affecting gene regulation and improving our knowledge of the pathogenesis of complex diseases. Despite the enormous amount of data regarding epigenetic modifications present in DNA and histones, deciphering their biological relevance in the context of the disease and health is currently still an ongoing process. Here, we consider the relationship between epigenetic research in tumorigenesis and the prospect of knowledge transfer to clinical use, focusing primarily on the epigenetic histone post-translational modifications, which could be used as biomarkers. We additionally focus on the use of proteomic techniques in research and evaluate their usefulness in clinical setting. KEYWORDS: cancer . diagnostics . DNA methylation . histone modification . immunohistochemistry . mass spectrometry

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post-translational modifications

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translation

Epigenetics is a rapidly expanding field in evolution, biology and medicine, exerting a complex gene-environment modulation of gene transcription, heterochromatic gene silencing and genome stability. Epigenetic modifications control different biological processes such as cell differentiation, proliferation, pre-mRNA processing, survival, genomic imprinting, nucleosome remodeling and X chromosome inactivation. They are also gaining recognition as a mechanism that enables translation of environmental factors into short-term or long-term heritable epigenetic marks [1–6]. From the beginning of the 20th century, the definition of ‘epigenetics’ has evolved to include any mechanism that alters gene expression without changing the DNA sequence; and some, but not all, epigenetic modifications are inheritable [7]. Three distinct and intertwined epigenetic mechanisms are DNA methylation and hydroxymethylation, post-translational modifications (PTMs) of histone proteins that include methylation, acetylation, ubiquitination, sumoylation, phosphorylation, ADP-ribosylation, proline isomerization, citrullination, butyrylation, propionylation and glycosylation

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validation

and regulation by small and large non-coding RNAs (ncRNAs) [1,8,9]. The epigenetic code is written in enzymatic modifications of cytosine bases and histone proteins in nucleosomes, which together with other proteins constitute chromatin [7]. Chromatin is a highly ordered structure, which is constantly modified and altered in several layers [8]. Its basic unit is nucleosome, in which 145–147 base pairs of DNA are wrapped around an octamer of core histones comprised of two copies of each of H2A, H2B, H3 and H4 proteins [5]. The linker histone H1 and its isoforms at the base of nucleosomes are involved in nucleosome compaction [10]. Each core octamer with wrapped DNA provides structural stability and capacity to regulate gene expression, depending on modifications of N-terminal histone tails, which are disordered and flexible [11]. Covalent modifications, especially methylations, are not confined only to these protruding tails, but are also found throughout the structured globular regions of histones (FIGURE 1) [5]. Epigenetic marks on histones and DNA are established, read and erased by a set of specific proteins [11]. There is also significant cross-talk between all

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ˇ Paska & Komel Hudler, Videtic

Transcriptional activation Transcriptional repression H2A

Ac

Me2

Me2 R

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Unknown role

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Transcriptional activation Transcriptional repression H3

Transcriptional activation

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K 12

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K 91

Figure 1. Major post-translational modifications of core histones with proposed biological role in transcription regulation. Ac: Acetylation; Me1: Monomethylation; Me2: Dimethylation; Me3: Trimethylation.

epigenetic mechanisms, namely, between DNA methylation, histone modifications and ncRNAs, enabling these highly regulated patterns to define the transcriptional activity of chromatin, either repressing or enabling access of transcriptional machinery to DNA sequence [11]. Epigenetic marks have the direct influence on the chromatin structure, which in turn affects transcript stability, DNA folding, nucleosome positioning, chromatin compaction and pre-mRNA splicing [2,10]. Epigenetic modifications are normally tightly controlled in the cell; however, emerging evidence showed that they can be influenced by several factors, including aging, environment, lifestyle, drugs and disease [6]. Indeed, research advances in recent years strongly implicate that dysregulated epigenetic modifications alone or in conjunction with genetic alterations could be driving the initiation and development of several complex diseases [6,9]. Epigenetic changes, including histone acetylation, histone methylation, alterations in ncRNA expression, and DNA methylation are now thought to play important roles in the onset and progression of cancer in numerous tumor types [5,8,12–14]. The interplay between histone PTMs, DNA 198

methylation and ncRNA expression alterations is diverse, complex and far from fully understood. Furthermore, physiological cell states define and lead these complex cross-talks in conjunction with all gene expression regulation systems in the cell as well as other signals from the body and the environment. Nevertheless, diverse aberrant epigenetic mechanisms have been, in addition to cancer, also associated with other diseases, such as diabetes, obesity, neurological disorders, asthma etc. [15–19]. The rapidly expanding knowledge of epigenetics is likewise challenging and revising traditional paradigms of potential inheritance [7,20–22]. Several research projects showed that nutrients and exposure to different environmental stressors additionally influence the epigenome [6,23–25]. Furthermore, the age-dependent variability of epigenetic marks was observed in the studies of monozygotic twins [26,27]. Epigenetic marks, established early in the life due to different factors, can become trans-generational and can be transmitted through several generations [6,24]. In this regard, we can consider the dynamic epigenome as the interface between environmental exposures and more stable genetic information stored in the genome Expert Rev. Proteomics 12(2), (2015)


Proteomic strategies for epigenetics

sequence [6,25]. Inheritance of epigenome through many generations allows the organisms in changing environment a quick mode of adaptation in contrast to slower establishment of changes in the DNA [24]. Yet another important aspect of epigenomic complexity is that, in contrast with stable genetic modifications, epigenetic changes are readily reversible through the action of so-called epigenetic readers, writers and/or erasers and even with certain drugs [6]. The plasticity of epigenetic modifications and reversibility of aberrant epigenetic changes have therefore emerged as a potential strategy for the development of new treatments of complex diseases [1]. Functional implications of epigenetic marks are being extensively studied as it is becoming clear that impairment of epigenetic memory initiates pathobiological processes. For example, cancer initiation and progression are regulated by both genetic and epigenetic events [28]. Global DNA hypomethylation and promoter-specific hypermethylation are often detected in different types of cancers and early-stage tumors [1,29,30]. Unfortunately, it is still little known how the epigenetic modifications are regulated [6]. Cis-acting sequence regions, containing certain polymorphisms, could influence the epigenetic landscape in specific cell types. This suggests that epigenetic alterations, including DNA methylation, histone modifications and ncRNAs, acting in concert with existing polymorphisms in the DNA, could be early initiators of tumorigenesis. The consequences of such orchestrated events at the level of dynamic epigenome and DNA sequence elements still need to be elucidated. The purpose of this review is to establish the relationship between enormous amounts of research data on epigenetic histone modifications in common cancers, as well as the prospect of knowledge transfer to clinical use with emphasis on the applicability of histone PTMs as biomarkers indicating the development, progression and recurrence of cancer. We focused on the use of proteomic methods in research and evaluated the usefulness of genomic and proteomic techniques in the clinical setting. In addition, we discussed the applicability of current proteomic methods and the need for establishment of rigorous validation tests for diagnostic purposes. Proteomic approaches for epigenetic studies

Proteomic-based analyses are gaining a more central role in the research of epigenetic biomarkers. Namely, proteomic studies contribute to the completion of the picture of more real-time reflection of cell processes that have already been extensively described by the RNA expression studies. It is widely accepted that mRNA concentrations do not always correspond to the protein levels in the cell, while the proteins represent the connection between RNA levels and physiological status of the cell. Furthermore, protein PTMs also significantly contribute to the increasing complexity of the proteome in comparison to the genome. Due to ascertainment of these facts and with quick technical advancements, proteomic approaches for the identification of biomarkers evolved rapidly in the past decade. Proteomic biomarkers with their PTMs are gaining importance informahealthcare.com

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and are becoming relevant biomarkers for detection, and prevention, and eventually they could also be suitable targets for developing novel treatment approaches for different cancers [31,32]. However, it should be noted that despite enormous efforts of several research groups, so far, none of the histone PTMs has been efficiently translated as epimarker into routine diagnostic laboratories, mainly due to the complexity of different histone PTMs and lack of thorough elucidation of their biological role. Histone PTMs can be readily interrogated with most traditional and novel high-throughput proteomic techniques. In general, proteomic approaches can be divided whether the proteomic methods are knowledge-based or discovery-driven (TABLE 1). Among the first mentioned are methods, such as ELISA, immunohistochemistry (IHC), immunoprecipitation methods, tissue microarray (TMA), Western blotting and protein and reverse-phase protein arrays [33]. Use of these techniques demands prior knowledge on proteins as well as their PTMs, and they are based on specific binding of antibodies to their targets. The most significant drawback of these methods is the availability of specific antibodies and also rather limited number of samples that can be tested, with the exception of TMA, which enables simultaneous analysis of up to 1000 separate tissue cores and is today used for research as well as for diagnostic purposes [32]. This upscaling reduces manual labor and costs [34,35]. Similarly, more samples can be tested with the use of reverse phase protein array, while more markers can be interrogated with protein arrays [32]. Although we can, in a way, circumvent the high-throughput issue with the use of the latter three methods, we still cannot get any information on the co-occupancy of the PTMs [36], and no novel data on proteins and their PTMs can be obtained. However, a contemporary discovery-driven method that does not require extensive information on interrogated proteins, but is actually used for determination of new protein biomarkers, PTMs and the co-localization of these, is mass spectrometry (MS). Many different variations of this technology exist, and they are all based on measuring mass-to-charge ratio of the peptides in the gas phase [37]. In epigenetic studies, the MS technology has been used for distinguishing histone variants; elucidating, mapping and determining combinations of histone modifications; and the discovery of histone-binding proteins, therefore, in short, for the determination of histone code [31,38]. The MS instrument is comprised of a sample inlet, an ion source, a mass analyzer and one or more detectors. There are several different mass analyzers, like quadrupole, ion trap, TOF and Fourier transform ion cyclotron resonance [39]. The ionization of proteins can be achieved by two different techniques: electrospray ionization, where the peptides in a liquid phase are ionized into a gas phase, or matrix-assisted laser desorption/ionization, where peptides are generated from solid phase. When the peptides in the analysis have the same mass, another fragmentation of those isomers is required in order to assure unequivocal identification. This is performed with collisioninduced dissociation, electron transfer dissociation or electron 199

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Table 1. Features of selected proteomic methods. Knowledge-based proteomic methods

Advantages

Disadvantages

ELISA

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Quantitative, robust, specific and simple for use No sophisticated instrumentation required Simultaneous analysis of a relatively large number of samples

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High throughput – analysis of up to 1000 samples at once (cost-effective) Consistent conditions for all the samples included in one array Use of only a small portion of sample, therefore it can be used for many analyses Useful for archival tissue samples Possible automation

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Simultaneous determination of a great range of analytes (high throughput) Small sample amounts Easy to use Great variety of different arrays increases experiment flexibility Automatization

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Large number of samples tested in one setting (high throughput) Small sample amounts Quantitative (also for PTMs) when high-quality antibodies are used

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Used for low-abundance proteins Direct quantification and very specific High-throughput sample analysis No need for specific antibodies

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IHC/TMA

. . . . .

Protein array

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Reverse-phase protein array

. . .

SRM–MS

. . . .

. .

. . .

. .

. .

. .

Need for specific antibodies Careful selection of primary in secondary antibodies to avoid cross-reactivity is necessary Optimization of procedure (washing, etc.) Need for specific and validated antibodies Need for good-quality tissue Standardized laboratory techniques with a skilled technician One analyte (one PTM) per reaction

Not possible to prepare the array in laboratories, since production is time consuming, labor-intensive and costly No standardized guidelines for experiment execution and data analysis Limited to antibody availability

Need for high-quality of monospecific antibodies Standardization and reproducibility of the experiment is still needed Limited sensitivity to detect low abundance proteins

Applicable only for known proteins and PTMs in a protein mixture Need for selection of peptide signature for target protein Insufficient sensitivity

Discovery-driven proteomic methods Top-down MS/MS

. . .

Bottom-up MS/MS

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Monitoring of complex mixture of proteins generating global view Information on co-occupancy and hierarchy of PTMs Complete protein sequence coverage (identification of isoforms)

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Monitoring complex spectrum of PTMs within single protein

.

. .

. .

Not high throughput Not for hydrophobic proteins or higher molecular mass (>100 kDa) proteins Not applicable for very small sample amounts (often the issue in case of clinical samples)

Need for pre-existing database on proteins Generally, not entire sequence of a protein is detected Lack of PTM coordination

IHC: Immunohistochemistry; MS: Mass spectrometry; PTM: Post-translational modifications; SRM–MS: Selected reaction monitoring mass spectrometry; TMA: Tissue micro array.

capture dissociation. In these cases, where two MS spectra need to be obtained for the identification, the technique is termed tandem MS/MS [37,38]. The use of specific combination of sample separation prior to the analysis, ionization procedure and detectors depends on the nature of the sample and on the purpose of the study. There are three different approaches in MS-based proteomics for the identification and quantification of proteins and their 200

PTMs: ‘bottom-up’, ‘top-down’ and ‘middle-down’. Proteolytic digestion of protein mixture into short fragments with subsequent MS analysis is designated as ‘bottom-up’ proteomics. After the extraction of proteins, these are subjected to trypsin digestion, which occurs at the C-terminals of lysine and arginine residues. Since the N-terminal ends of histones are rich in both lysine and arginine, they need to be derivatized with propionic anhydride, which blocks trypsin cleavage of both Expert Rev. Proteomics 12(2), (2015)



unmodified residues and also mono-methylated lysine, prior to the treatment with the enzyme. Derivatization step is important from two aspects: it prevents production of peptides too short for MS analysis and increases the hydrophobicity of histone peptides, which increases chromatography resolution [37,38]. The ‘bottom-up’ approach is convenient if only confirmation of protein presence with the identification from a database is required [39]. The contrast of the ‘bottom-up’ strategy is the ‘top-down’ strategy, which gives the global overview of proteins and their PTMs. In this approach, the proteins are not digested, they are rather kept intact. Probably the most critical step in this case is effective and high-throughput chromatographic separation of proteins before they are introduced into the instrument. For the fragmentation electron transfer dissociation or electron capture dissociation is used, since the histone proteins are highly charged and have numerous PTMs, while in the case of ‘bottom-up’ approach high-energy collision-induced dissociation, which can cause loss of liable PTMs, is used [37,38]. The most valuable information obtained by the ‘top-down’ approach is the data on PTMs, their co-occupancy and hierarchies [40], and also on their abundance [41]. The ‘middle-down’ approach is applied when larger peptides, up to about 50 amino acid residues (4–7 kDa), are studied. In the epigenetic studies, this means that N-terminal ends, which protrude from the histone proteins and carry the most PTMs, are being analyzed [7,42]. In this strategy, enzymes, like Glu-C, which cleave adjacent to less abundant amino acid residues in the histone proteins resulting in longer peptides, are used. Otherwise, the procedure is similar to the ‘top-down’ approach [38]. The ‘top-down’ MS approach has so far proved to be successful in providing a global overview of protein forms, their abundances and detection of PTMs [40]. However, it is not yet ready to be used for high-throughput proteomics [31,38,39], for the characterization of proteins with higher molecular mass (>100 kDa) and highly hydrophobic proteins [41]. Today, most of the work is still performed with the ‘bottom-up’ approach [38], and several challenges in the analyses, like co-elution of nearly isobaric peptides, high abundance of the same PTMs in near proximity but in different arrangements, are still being addressed [43]. Due to different advantages and drawbacks of both approaches, they seem to complement each other in the integrated strategies for protein analysis. In cancer epigenetics, the most often studied histone PTMs are acetylation and methylation, which we will discuss in the following sections of the paper. Much less attention has been turned toward other PTMs; however, interested readers can find the list of most recently identified PTMs of the core nucleosome histones in the review by Arnaudo and Garcia [44]. Translation of proteomic research into routine clinical laboratory applications

Throughout the world, we see more and more examples of how modern genetic and proteomic research translate into diagnostic and therapeutic fields of clinical settings, leading to personalized medicine [45,46]. In the last few decades, important informahealthcare.com

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advances have been made in the area of classical and highthroughput genomic technologies, which have been successfully translated into diagnostic assays for improving diagnosis, prognosis and management of a variety of diseases (TABLE 2) [7,47,48]. From the genomic point of view, DNA methylation is the most often studied epigenetic modification. In the context of epigenetic dysregulation, the genetic testing for hypermethylation of MLH1 and MSH2 promoter has become an established method in diagnosis of sporadic colorectal cancer (CRC) [49–51]. A selected number of laboratories are also implementing the detection of MGMT methylation in diagnostics of glioma [52]. With the improvement of novel molecular genetic techniques, such as next-generation sequencing, several previously labor-demanding techniques have become much faster, more versatile in their applications and affordable even in routine multiplex and large-scale genome interrogations. On the other hand, whole genome analyses of histone modifications with preceding chromatin immunoprecipitation (ChIP) and subsequent use of microarrays or direct next-generation sequencing are still too expensive for wider use. Currently, their main applicability is restricted to research-based mapping of different histone modifications in diseases. In addition to the relative high costs, the other important drawback of conventional ChIP assays represents the requirement of a large numbers of cells. This is particularly pronounced when rare and small tissue or cell samples come into consideration and/or when lengthy procedures are applied [7]. Similarly, MS-based proteomics is indispensable in research and discovery of potential biomarkers; however, the technology is relatively slowly assimilating into clinical laboratory, mainly due to its high cost, the need for training the personnel, the complexity of data analyses and the difficulties associated with sensitivity and specificity of tests [48,53–55]. On the other hand, commercially available reliable proteomic testing is paving its way into personalized diagnostics [56]. For example, OncoPlexDx is among other cancer proteomic panels offering a MS-based selected reaction monitoring (SRM) assay for the EGFR utilizing Liquid Tissue-SRM technology platform (EGFR–SRM). The test was evaluated on a collection of histologically characterized paraffin-embedded tumor tissues of patients with non-small cell lung cancer. The added value of this research is also analytical validation of EGFR protein levels in a collection of fresh and paraffin-embedded tissue culture cell lines with EGFR–SRM method and ELISA sandwich immunoassay. The method was further validated on xenograft tumor samples, which were previously characterized by IHC of formalin-fixed tissue and Western blot analysis of frozen matching tumor tissue [56]. Hembrough et al. showed that comparable results were obtained between formalin-fixed and matching frozen tissue using MS-based approach, and they demonstrated its accurate specificity and sensitivity. They also concluded that EGFR–SRM assay is a highly promising approach to quantitating and monitoring EGFR levels in patient tumor tissue applicable to identification of patients who would benefit from EGFR-inhibitor therapies [56]. Another successful story 201

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Table 2. Selection of representative FDA-approved protein biomarkers. Biomarker

Cancer type

Specimen

Methodology

Commentary (intended use)

CA15-3

Breast

Serum, plasma

Immunoassay

Used as an aid in the monitoring disease progression, response to therapy and in the detection of recurrence in previously treated stage II and III breast cancer patients

CA19-9

Pancreatic

Serum, plasma

Immunoassay

Used as an aid in the monitoring disease progression and response to therapy

CA27.29

Breast

Serum

Immunoassay

Used as an aid in monitoring patients previously treated for stage II or Ill breast cancer, monitoring disease response to therapy

CEA

Breast, colon, pancreatic

Serum, plasma

Immunoassay

Indicated as an aid in the monitoring of cancer patients in whom changing concentrations of CEA are observed

Circulating tumor cells (EpCAM, CD45, cytokeratins 8, 18+, 19+)

Breast, colorectal

Whole blood

Immunomagnetic capture/ immunofluorescence

Used an aid in the monitoring patients with metastatic breast or metastatic colorectal cancer; the number of circulating tumor cells allows assessment of patient prognosis and is predictive of progression-free survival and overall survival

ER

Breast

FFPE tissue

Immunohistochemistry

Used as an aid in the management, prognosis and prediction of hormone therapy for breast carcinoma

Fibrin/fibrinogen degradation product (DR-70)

Colorectal

Serum

Immunoassay

Used as an aid in monitoring the progression of disease after therapy

HER-2/neu

Breast

FFPE tissue


Used as an aid in the assessment of breast cancer patients for whom Herceptin treatment is considered

Human hemoglobin (fecal occult blood)

Colorectal

Feces

Immunoassay

Recommended for screening for CRC or gastrointestinal bleeding from any source

PR

Breast

FFPE tissue


Indicated as an aid in identifying patients eligible for endocrine treatment, as well as an aid in the prognosis and management of breast cancer

CEA: Carcinoembryogenic antigen; ER: Estrogen receptor; FFPE: Formalin-fixed and paraffin-embedded; PR: Progesterone receptor.

regarding implementation of proteomic diagnostic test is the development of OVA1 test, which was in 2009 cleared by the US FDA [57]. The specificity and sensitivity of this test has been validated in several large studies [57–59]. Interestingly, although initially the panel of biomarkers for the evaluation of ovarian tumor mass was identified using SELDI–TOF MS platform, the commercially available test was developed as blood-based immunoassay, because of better clinical and analytical performance [57]. It is important to note that none such thorough study has been performed on histone PTMs. Next, IHC represents a more simplistic and robust approach to detect specific antigens based on antigen–antibody interactions [35,38]. IHC has already been used in routine diagnostics, therapeutic decision making and disease prognosis for a few decades and is a method of choice for localization and visualization of proteins in tissues or cells (immunocytochemistry) [60]. Laboratories usually establish their own tests; however, only a few tests have been cross-standardized and cross-validated 202

among different laboratories; thus the main drawback of IHC is lack of reproducibility across different clinical laboratories due to different methodologies used, different modes of tissue preparation and storage, different antibodies, diverse detection systems and interpretation issues [60]. Cross-validation and cross-standardization are common issues encountered in the process of translation of research knowledge based on almost all proteomic and genomic techniques into diagnostic laboratory setting. As the epigenetic field is gaining more and more recognition in cancer pathogenesis, researchers and companies produced and are still producing antibodies that specifically recognize the core histones and their variants modified by lysine acetylation, biotinylation, mono-, di- or tri-methylation; arginine mono- or di-methylation; serine or threonine phosphorylation and arginine substitution with citrulline. Several of these antibodies are currently being used and evaluated in different research studies. However, before assays can be translated into routine clinical laboratories, they must be thoroughly Expert Rev. Proteomics 12(2), (2015)



validated in large research clinical studies. Several groups have already attempted to validate the sensitivity, specificity, crossreactivity and affinity of PTM antibodies, and we refer the interested reader to these valuable studies [61–64]. In conclusion, as the traditional protein methods, such as IHC and ELISA, have already been firmly implemented into diagnostic laboratories, it is realistic to expect that knowledge on epimarkers, obtained from novel, mostly discovery-driven proteomic methods, will be translated into more simple immunoassays. However, advances in proteomic techniques are enabling more specific and accurate determination of proteins and MS platforms are slowly but surely progressing into clinical setting, primarily through privately owned companies offering some of the novel diagnostic proteomic tests. Proteomic approaches for epigenetic studies in clinical research of various cancers

The term clinical research in this manuscript encompasses all research, which involves clinical tissue samples obtained from patients, with the aim to decipher deregulated molecular pathways and gain data on molecules or modifications of molecules, which could be used as biomarkers. The primary focus of this review is to present current research findings in the field of deregulated histone PTMs in selected types of gastrointestinal cancers and breast cancer. As discussed previously, the most often used proteomic method in clinical use is IHC, since it is reliable, fast and, due to relative high throughput with the use of TMA technology, also cost-effective. However, as the field of epigenetics is still young, the discovery phase of suitable biomarkers requires high-throughput methods, which could perform global identification of the epigenetic landscape in diseased subjects in order to pinpoint the most suitable panel of biomarkers for the detection of specific cancers. In this regard, biomarkers that would be uniformly applicable in different laboratories and informative regarding the diagnosis, treatment and prognosis as well as several novel proteomic approaches, such as MS, ChIP and so on that are being used in the analysis of epigenetic markers are being pursued. Unfortunately, given the differences between different research approaches, a significant bottleneck persists in the comparability across the studies. On the other hand, high-throughput proteomic methods are the key generator of novel data on the epigenetic biomarkers, opening several novel biological questions [6,65]. The most pressing issue in carcinogenesis is related to difficulties in interpreting the biological relevance of epigenetic marks that were found to be dysregulated in cancer [6,65]. In the following section, we discussed recent discoveries regarding histone modifications in common solid cancers, which were selected considering their high mortality rates, heterogeneous nature and the fact that they share some of the most common histone PTMs alterations (TABLE 3). Breast cancer

Breast cancer is a heterogeneous disease, divided into multiple subclasses which differ in risk factors and also clinical outcome. informahealthcare.com

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Currently, its molecular classification and prognostic assessment is mainly based on immunostaining for several protein biomarkers and gene-expression profiling. So far, the proteomic studies of histone modifications did not find any suitable biomarkers that would represent a powerful tool for diagnostic, prognostic or predictive test for clinical use [47]. Histone modification status, particularly acetylation and methylation, has been extensively studied in the epigenetic profiling of breast cancer. The most recent study in this field has been published by Healey and co-workers, where in a prospective study designated as Nurses’ Health Study from 1976 to 2000, more than 120,000 registered female nurses between ages 30 and 55 years were enrolled. With TMA technique, they determined the global status of two epigenetic markers: trimethylation of histone H3 lysine 9 (H3K9me3) and lysine 27 (H3K27me3) on 804 cases of invasive breast cancer. They determined that methylation of both lysine residues overlapped substantially. For H3K27me3, results showed significant association with lower grade tumors and with estrogen and progesterone receptor-positive tumors, while it was inversely associated with EGFR-positive tumors. When other breast cancer risk factors were taken into consideration and were adjusted for, this particular epigenetic marker showed statistically significant association with luminal A subtype. Based on these results, they were able to conclude that increased levels of H3K27me3 are not the consequence of aneuploidy or global increase of H3, but represent an independent marker. The H3K9me3 was associated only with tumor grade [66]. The abundance of H3K27me3 has been analyzed on another set of wellcharacterized breast tumors and breast cancer cell lines. Similarly, as in the previously mentioned study, H3K27me3 had significantly different expression among the breast cancer subtypes and was associated with the luminal A subtype. Additionally, the expression analysis of enhancer of zeste homolog 2 (EZH2), which mediates the trimethylation of H3K27 and has been associated with aggressive phenotype in breast cancer, was performed. The results showed that the two markers do not necessarily correlate; therefore, it was speculated that EZH2 might have other effects. In the survival analysis, high levels of EZH2 and low levels of H3K27me3 were associated with poor survival outcome [67]. Somewhat more comprehensive study of epigenetic markers assessed global patterns of seven histone modifications: four lysine acetylations (H3K9ac, H3K18ac, H4K12ac and H4K16ac), two lysine methylations (H3K4me2 and H4K20me3) and arginine methylation (H4R3me2) in association with biological and clinical factors on a set of well-characterized primary breast carcinomas. With TMA, it was determined that global levels of the histone marks varied among different tumors, which could be associated to their differences in biology, clinicopathological features and prognostic factors. Low levels of all seven histone marks were associated with high tumor grade. Poor prognostic subtypes and adverse patient outcome were also related to low level of detection of the tested epigenetic changes. However, high levels of global markers, which were associated with a 203

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Table 3. Putative epigenetic histone biomarkers in selected cancer tissues. Histone epigenetic mark

Cancer type

Method

Type and number of samples

Main results (association with clinical data, known markers, etc.)

Ref.

H3K4me2

Colorectal cancer

IHC, ChIP/WB

80 patients with CRC, different histological stages

Higher-score cases of H3K4me2 tended to exhibit deeper tumor invasion and an advanced pathological stage compared with lower-score cases

[92]

Pancreatic cancer

IHC/TMA

Pancreatic adenocarcinoma from two cohorts: 195 patients with adjuvant gemcitabine or fluorouracil and 140 patients with stage I and II disease

Lower levels of methylation associated with worse survival outcomes

[72]

IHC/TMA

Pancreatic adenocarcinoma from 61 patients; gemcitabine treatment offered

Low expression associated with presence of perineural invasion, elevated serum CA19-9, and worse disease-free survival in patients with gemcitabine therapy High expression associated with smaller tumor size

[74]

Breast cancer

IHC/TMA

880 breast tumors

Low level of histone marks associated with high tumor grade

[68]

Colorectal cancer

IHC

254 patients with CRC, TNM stage I-III tumors

Low nuclear expression of histone mark was associated with good prognosis

[94]

Pancreatic cancer

IHC/TMA


Higher expression related to well and moderately differentiated tumor

[74]

Colorectal cancer

IHC

89 patients with CRC, different histological stages

Dimethylation of histone mark was higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells

[93]

IHC/TMA

Pancreatic adenocarcinoma from two cohorts: 195 patients with adjuvant gemcitabine or fluorouracil and 140 patients with stage I and II


[72]

IHC/TMA


Low expression associated with poorly differentiated adenocarcinomas or histological types other than adenocarcinoma

[74]

Gastric cancer

IHC/TMA

261 patients with gastric adenocarcinoma

Trimethylation of histone mark positively correlated with tumor stage, lymphovascular invasion, cancer recurrence, and poor survival rate

[12]

Colorectal cancer

IHC/TMA

254 patients with CRC, TNM stage I–III tumors

High expression of histone mark was associated with good prognosis

[94]

Breast cancer

IHC/TMA

804 cases of invasive breast cancer

Associated with tumor grade

[66]

Gastric cancer

ChIP-chip HIP–RTPCR

Eight patients with gastric adenocarcinoma

One hundred twenty-eight (119 increased and 9 decreased H3K27me3) genes displaying significant H3K27me3 differences were found between tumor and adjacent non-tumor tissues

[83]

Pancreatic cancer

IHC/TMA

Pancreatic adenocarcinoma from 165 patients + 72 normal samples adjacent to cancer

Associated with tumor grade. 5-year survival for low expression 11%, for high expression 23%

[73]

H3K4me3

H3K9me2

Pancreatic cancer

H3K9me3

H3K27me3

CRC: Colorectal cancer; ChIP: Chromatin immunoprecipitation; ER: Estrogen receptor; IHC: Immunohistochemistry; PR: Progesterone receptor; TMA: Tissue micro array; WB: Western blot.

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Table 3. Putative epigenetic histone biomarkers in selected cancer tissues (cont.).


Histone epigenetic mark

Ref.

Cancer type

Method



Breast cancer

IHC/TMA

804 cases of invasive breast cancer

Associated with lower tumor grade, and putatively with estrogen receptor-positive tumors. Associated with luminal A subtype

[66]

IHC/TMA

237 tumor form node negative patients and 564 tumors form premenopausal women

High methylation level present in luminal A, HER-enriched and normal-like tumors. It was associated with better disease-free survival and small tumor size Low levels associated with ER/PR negative and histological grade 3 tumors

[67]

IHC/TMA

Breast cancer form 142 patients and 43 adjacent non-tumor samples

Low expression associated with large tumor size, ER negative, lymph node positive 5-year survival for low expression 46, and 72% among the rest

[73]

H3ac

Colorectal cancer

IHC, ChIP/WB

80 patients with CRC, different histological types

Moderately/poorly differentiated histological types, advanced pathological stages (Stage III/IV) and high expression of H3Ac predicted poor overall survival

[92]

H3K9ac

Pancreatic cancer

IHC


Low expression associated with absence of lymph node metastasis

[74]

Breast cancer

IHC/TMA

880 breast tumors of different histological types

Low detection level associated with large tumor size and high tumor grade. High level of histone mark associated with low lymph node stage and longer disease-free survival

[68]

Gastric cancer

IHC/TMA


Acetylation of histone mark was associated with poorly differentiated or diffuse type histology

[12]

Pancreatic cancer

IHC/TMA

Pancreatic adenocarcinoma from two cohorts: 195 patients with adjuvant gemcitabine or fluorouracil and 140 patients with stage I and II


[72]

IHC/TMA


No association determined

[74]

Breast cancer

IHC/TMA


Low level of histone mark associated with high tumor grade. High level of histone mark associated with longer disease-free survival

[68]

H4K12ac

Breast cancer

IHC/TMA


Low detection level associated with high tumor grade

[68]

H4K16ac

Gastric cancer

IHC/TMA

261 patients with gastric cancer

Cases with acetylated histone mark had better prognosis than cases with no acetylation, the difference was not statistically significant

[12]

Colorectal cancer

IHC/TMA

254 patients with CRC

Acetylation of histone mark showed lower nuclear expression in tumor samples than in non-tumor samples

[89]

Breast cancer

IHC/TMA


Low detection level associated with large tumor size, high tumor grade, and vascular invasion

[68]

H3K18ac



205

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Table 3. Putative epigenetic histone biomarkers in selected cancer tissues (cont.). Histone epigenetic mark

Cancer type

Method



Ref.

Low or absent marker could represent early sign of breast cancer


H4K20me3

H4R3me2

Gastric cancer

IHC/TMA


No association determined

[12]

Colorectal cancer

IHC/TMA

254 patients with CRC, TNM stage I-III tumors

Higher levels of methylated histone mark were associated with good prognosis

[94]

Breast cancer

IHC/TMA


Low detection level associated with high tumor grade

[68]

Breast cancer

IHC/TMA


Low detection level associated with large tumor size and high tumor grade. High-level of histone mark associated with low lymph node stage and longer disease-free survival

[68]


favorable prognosis, were predominantly (93%) detected in luminal-like tumors. In the histone cluster analysis, they were able to clearly identify three groups of histone modification patterns with distinct correlations to prognostic features and clinical outcome [68]. LeRoy and coworkers performed a ‘bottom-up’ MS to prepare an atlas of histone modification signatures from 24 different human cell lines, two of which were from breast carcinoma (MCF7 and MDA-MB231). Although the two investigated breast cancer cell lines originate from very different breast cancer molecular subtypes, they had similar chromatin modification patterns, while their PTM profiles differed from profiles of cell lines from other tissue types. The findings presented diverged also from results of another study [67], and the authors argued that this could be due to the different methodologies (IHC vs MS) applied in both studies. Besides proteomic classification, they also analyzed genetic background of histone-modifying enzymes in the tested cell lines and showed consistent classification across both approaches. Building of such an atlas, where 37 unique histone modification patterns on histone H3 and 19 on histone H4 were determined, represents an important resource for directing the investigations of epigenetic markers for analysis and classification of cancer cells [69]. Pancreatic cancer

One of the most aggressive and lethal malignancies is pancreatic cancer. Since the disease symptoms are of rather unspecific nature, it is often diagnosed in advanced stages. With current diagnostic approaches, accurate differential diagnosis as well as timely detection is extremely difficult. Although usually more than one method (e.g., imaging, and histopathological or cytopathological assessment) is applied, none of them is specific and sensitive enough [70]. 206

In the detection of pancreatic ductal adenocarcinoma, the carbohydrate antigen (CA) 19-9 is considered as the gold biomarker standard. However, since it has low sensitivity and specificity, it is not most suitable for clinical application [71]. Therefore, the search for accurate epigenetic biomarker(s) is needed to achieve more accurate prognosis. Results of two studies on Caucasian population performed by immunohistochemical staining showed that low cellular levels of dimethylation of histone H3 lysine 4 (H3K4me2) and lysine 9 (H3K9me2) and trimethylation of lysine 27 (H3K27me3), as well as histone H3 lysine 18 acetylation (H3K18ac) represented independent and significant predictor for poor patient survival [72,73]. Particularly, lower cellular levels of H3K4me2 and H3K9me2 were found to be associated with worse survival outcomes for patients on fluorouracil, but not gemcitabine adjuvant chemotherapy [72]. Watanabe and coworkers examined H3K4me2 and trimethylation of histone H3 lysine 4 (H3K4me3), as well as H3K9me2 and H3K9ac in Japanese population and were, in contrast to the two previously mentioned studies, not able to show association with patient survival [74]. However, in the group of patients, who completed adjuvant gemcitabine treatment, they found low levels of H3K4me2 to be statistically significantly associated with worse disease-free survival. What was also interesting is that the group with low expression of H3K4me2 had significantly elevated serum CA 19-9 concentrations [74]. Based on the results of the three studies, we could conclude that determination of the H3K4me2 status could be an informative biomarker for the patients who would likely receive adjuvant gemcitabine or fluorouracil chemotherapy. An important finding on epigenetic silencing of tumor-suppressor genes, which play a relevant role in carcinogenesis, was shown by Kumagai and coworkers when studying different pancreatic cell lines. They pursued the histone acetylation status of the CEBPA gene and determined Expert Rev. Proteomics 12(2), (2015)


50-fold increase in signal intensity when pancreatic cell line PANC-1 was treated with histone deacetylase inhibitor suberoylanilide hydroxanic acid compared to non-treated cells. Moreover, aberrant localization of C/EBPa protein was determined by IHC, where in addition to lower signal in the nucleus, the presence of the signal was also detected in the cytoplasm [75].


Gastric cancer

Gastric cancer remains a global healthcare burden as the second leading cause of cancer-related deaths [12,76–78]. Despite several decades of extensive studies, the molecular pathways and genes involved in the disease development and progression are still not elucidated and the prognosis for patients with gastric cancer remains poor [79,80]. Recent data showed that multistep and heterogeneous onset of the gastric carcinogenesis is further complicated with epigenetic dysregulation [8,12]. Park et al. investigated the global modification patterns of H3K9, H4K16 and H4K20 in 261 gastric adenocarcinomas using IHC [12]. Although this approach did not reveal which specific genes were affected and how the gene activity was modulated, they were able to associate the level of H3K9 trimethylation (H3K9me3) with invasion, lymphovascular invasion, recurrence and survival. Interestingly, when they categorized patients into three groups, acetylation dominant, methylation dominant and co-dominant/negative, they found out that methylation group had the worst prognosis, whereas the acetylation group had the best prognosis [12]. They concluded that the pattern of PTMs detected by IHC could be used as a predictor of cancer recurrence and as an independent prognostic factor in gastric adenocarcinomas. Using ChIP–BSseq technology (ChIP followed by bisulfite sequencing), Gao et al. found that CpG islands, (± 500) transcription start sites and exons were preferentially enriched in H3K27me3 marks in two gastric cancer cell lines [81]. The DNA methylation levels were extremely low at these genomic regions enriched with H3K4me3 marks, indicating a positive correlation of geneexpression activity. They also compared histone modification patterns and DNA methylation patterns between lymphoblastoid cell line (YH), two gastric cell lines (BGC-823 and AGS), cervical cancer cells (HeLa), colon cancer (HCT116), prostate cancer (LNCaP) and normal prostate epithelial cell line (PrEC). Their analyses revealed variable patterns of both H3K27me3 marks and DNA methylation in different cell populations. Clustering analysis of the seven cell lines based on the average values of cytosine methylation levels in all promoter regions enriched with H3K27me3 marks showed two normal cell lines (YH and PrEC) were clustered together and separated from remaining five cancer cell lines. Thus, despite the distinct cellular lineages of these cell lines, the clustering results suggested that onco-epigenomic signatures of the DNA methylation pattern in the sequences bound to the H3K27me3 marks are different from normal epigenomic signatures. In addition, it was also found that Helicobacter pylori, a group I carcinogen for gastric cancer, alters histone informahealthcare.com

Review

modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms [82]. Using ChIP, it was determined that alterations in c-jun and hsp70 gene expression were associated with the H3S10 dephosphorylation. The researchers further noted that H. pylori induced a fast inhibition on H3S10 phosphorylation within 1 h. Zhang et al. attempted to characterize H3K27me3 modification in tumor and matched non-tumor tissues of gastric cancer patients [83]. Using ChIP-chip and ChIP–RT-PCR approaches, they confirmed that 128 genes displaying significant H3K27me3 differences were found between gastric cancer and adjacent non-tumor tissues, showing H3K27me3 could be used as potential biomarker. Although several of commonly occurring PTMs of histones were found in gastric cancer, the problem of detecting these changes in easily accessible diagnostic fluids, such as blood or urine, still persists. Colorectal cancer

CRC as one of the leading causes of cancer-related death results from an accumulation of genetic and epigenetic changes in colon epithelial cells, leading to their transformation into malignant phenotype [84]. The genetic basis of molecular mechanisms involved in the pathogenesis of certain types of CRC has been thoroughly researched in the last decades, and several models with initiating mutations leading to the development of CRC have been proposed [84–86]. For example, DNA methylation is well recognized as an important carcinogenic factor in the subgroup of sporadic CRC called CpG island methylator phenotype cancers [84]. The greatest progress has been made in our understanding of familial CRCs, leading to the establishment of several diagnostic and screening approaches [84,85]. However, these well-characterized hereditable CRCs account for only 10% of all CRC patients [85]. For another 10–25% of patients with hereditary type of CRC and for up to 80% of patients with sporadic CRC, the genetic predisposition and background of carcinogenesis remain unclear [84–86]. Genomewide profiling of histone modifications utilizing different proteomic approaches showed that epigenetic changes play an important role along with genetic alterations in the pathogenesis of this disease. By comparing histone modification patterns in normal mucosa, CRC tumors and cell lines, Enroth and coworkers discovered that both the H3K4me3 and H3K27me3 patterns were similar between the compared types of tissues but distinct compared to poorly differentiated CRC cell lines [87]. This raises the important question of how well the cell lines represent the actual changes in tissues of cancer patients. They identified four genes, KLF7, EBF3, DKFZp667I0324 and RBMS1, with altered expression between tumor and normal tissues due to differences in H3K4me3 and H3K27me3 marks. They also found several other changes in chromatin states around transcription start sites, which were different between examined patients, showing heterogeneity of epigenetic alterations. Several other recent publications reported on the methylation status of H3 methylation marks in various experimental settings [81,88–91]. 207


Review


The differences between their observations showed the complexity of epigenomic landscape, which still needs to be elucidated and placed in the context of all modifications occurring during pathogenesis. Several recent clinically relevant studies assessed the correlation of histone methylation and acetylation profiles with clinicopathological findings [89,92–95]. Immunohistochemical comparison of histone marks between tumor and non-tumor tissues showed significant differences in all studies. On the other hand, the findings regarding association of some of the examined histone marks and their impact on survival and prognosis tend to differ between studies. For example, combined analysis of H3K4me3, H3K9me3 and H3K20me3 showed that low levels of activating modification H3K4me3 and high levels of silencing modifications H3K9me3 and H4K20me3 were associated with good prognosis [94]. In another study, high H3K9me3 levels were associated with lymph node metastasis, implying worse prognosis [95]. Furthermore, it was shown that H3K9me2 modification is also more frequent in cancerous colon cells [93]. A novel study using bottom-up MS approach identified 96 modified peptides, where 41 distinct PTM sites were distributed among the amino-terminal tails and the globular domain of the four histones. Using this approach, it was shown that acetylation of H3K27 was upregulated in CRC tissues [96]. Expert commentary

Although we have seen tremendous advances in understanding chromatin biology and the development of suitable proteomic methods for epigenetic research, the direct translation into clinical setting is still far away. Integration of histone epigenetic biomarkers is hampered by the complexity of modifications and difficulties in deciphering their pathobiological impact on the disease. This is further complicated because different environmental stressors and lifestyle add another level of complexity to the epigenetic code. Thorough elucidation of the biological role of histone modifications and how the accompanying genetic mutations and aberrant expression patterns affect the pathogenic proteome in the disease is necessary. Furthermore, it is evident that comprehensive research efforts are required in order to decipher epigenetic marks in different cell lines and compare them to corresponding tumor tissues, since in most cases there are substantial discrepancies in histone PTM patterns between cancer tissues and cell models of the same type of cancer. Although cell lines are valuable models for deciphering signaling pathways, biological roles of proteins and so on, it should be noted that cells adapted to in vitro conditions invariably gain specific attributes distinct from their in vivo counterparts and lose some of the characteristics, typical for their in vivo relatives. Therefore, clinically valuable biomarkers will most likely stem from the research performed on tissue specimens, despite their histological and molecular heterogeneity. Another important challenge in the transfer of research knowledge into clinics is the fact that the majority of MS-based research focusing on PTMs of histones in solid carcinomas 208

mostly utilizes tumor tissues as clinical samples. With regard to the fact that epigenome is highly dynamic and influenced by different intrinsic and environmental factors, the research approach is justified; however, obtaining tissue biopsies is considered an invasive diagnostic method. The translation of tissue epigenome into the epigenomes of more easily accessible diagnostic samples, such as blood or urine, will again require additional research studies. Furthermore, generalization of research data across different studies is currently much hindered. Several studies also suffer from being underpowered and large multicentric studies could effectively overcome this limitation. However, careful planning of the studies and appreciation of interpopulation differences should be taken into consideration. Together with such large studies, it would also be prudent to build platforms supporting integration of research data from different research groups in order to perform more accurate bioinformatic studies involving more samples, thus increasing the power of the combined meta-analyses. On the other hand, IHC is already established in clinical setting as straightforward technique. With proper validation and standardization tests, it could be used for the detection of epigenetic modifications. However, aside from the determining the specificity and sensitivity of the epigenetic marks, in affinity-based technologies, the effect of SNPs and other genetic variations, which could be reflected in antigens, cannot be properly addressed. Introduction of more complex MS-based techniques could overcome these issues. Five-year view

MS is a powerful technique that enables a great variety of applications. Primary structure of peptides along with their PTMs can be determined with ‘bottom-up’ technique, while the ‘top-down’ approach provides the proteomic signature of a sample based on the analysis of intact proteins. In the past years, MS has proven to be the method of choice for identification of novel biomarkers, and also for spatial determination of PTMs co-occurrence. However, it has not been introduced into routine clinical laboratory work for diagnostics or screening, due to its limited throughput, and also still rather costly operations. Therefore, IHC is the workhorse in clinical laboratories, although it lacks the desired sensitivity and specificity. Similarly, as did the next-generation sequencing techniques advanced the genomic research, it could be expected that the MS-based approaches will speed up the proteomic studies when the technology will reach the stage of desired applicability. But until then, the focus of rather expensive MS studies of epigenetic makers should be oriented toward the discovery of epigenetic biomarkers that would be suitable for differential diagnosis and monitoring of patients and for targeted therapies. The most valuable application of the high-resolution ‘bottomup’ analysis could be the determination of epigenetic drugs efficiency, where data on very particular changes in PTMs that in turn affect the gene expression are analyzed. Expert Rev. Proteomics 12(2), (2015)



Another important issue that could be addressed in the next 5 years is the comparability across the studies. Namely, in cancer research, the majority of the studies currently provide data on clinicopathological parameters, histological and pathohistological status, phenotypic determination of particular cancer, outcome of treatment and survival. Although this seems to be very broad spectra of information, the comparability between the studies is quite limited. The differences in sample collection and storage, and also in laboratory procedures, are too pronounced to be able to produce comparable results and point out unambiguous biomarkers. This fact is rather depressing, since the patient samples are unique, limited in size and therefore of great value. Development of a procedure that would

Review

enable the development of diagnostic models covering all the steps from sample collection to objective bioinformatics analysis of data would greatly contribute to the translation of today’s exclusively research techniques into clinical practice. Financial & competing interests disclosure

This work was supported in part by the Slovenian Research Agency program grant No. P1-0104. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues .

Thorough elucidation of the biological role of histone modifications and discerning the influence of the accompanying genetic mutations and aberrant expression gene patterns is paramount to our understanding of carcinogenesis.

.

Despite years and years of search for cancer biomarkers, it has become apparent that only genetic changes cannot be sufficient to explain the molecular heterogeneity of cancer pathogenesis.

.

Epigenetic modifications are now thought to play an important role in the onset and progression of cancer in conjunction with genetic aberrations.

.

The heterogeneity of epigenetic marks in individual cancer patients should be addressed with regard to the impact of environmental factors and different types of treatments on epigenetic landscape.

.

The lack of integration of research on the DNA, RNA and epigenetic level is resulting in the fragmented knowledge. This incoherence is even more pronounced in the difficulties associated with translation of research into clinical setting.

.

Generalization of research data on epigenetic modifications across different studies is necessary in order to determine the best panels of epigenetic assays for cancer diagnostics.

.

Establishment of a networked framework/platform supporting integration of research data from different research groups is essential in order to perform more accurate bioinformatic studies involving more samples and to improve the power of the combined metaanalyses.

epigenetic control regions. Genome Biol 2012;13(10):R91

References Papers of special note have been highlighted as: . of interest .. of considerable interest 1.

2.

3.

4.

Ellis L, Atadja PW, Johnstone RW. Epigenetics in cancer: targeting chromatin modifications. Mol Cancer Ther 2009;8(6): 1409-20 Brown SJ, Stoilov P, Xing Y. Chromatin and epigenetic regulation of pre-mRNA processing. Human molecular genetics 2012 (R1):90-6 Manikkam M, Tracey R, Guerrero-Bosagna C, Skinner MK. Pesticide and insect repellent mixture (permethrin and DEET) induces epigenetic transgenerational inheritance of disease and sperm epimutations. Reprod Toxicol 2012;34(4):708-19 Skinner MK, Manikkam M, Haque MM, et al. Epigenetic transgenerational inheritance of somatic transcriptomes and


5.

6.

Li KK, Luo C, Wang D, et al. Chemical and biochemical approaches in the study of histone methylation and demethylation. Med Res Rev 2012;32(4):815-67 Handel AE, Ebers GC, Ramagopalan SV. Epigenetics: molecular mechanisms and implications for disease. Trends Mol Med 2010;16(1):7-16

.

A comprehensive review of epigenetic mechanisms in the context of environment, medicine, evolution, treatment, disease and health.

7.

Han Y, Garcia BA. Combining genomic and proteomic approaches for epigenetics research. Epigenomics 2013;5(4):439-52

8.

Kang C, Song JJ, Lee J, Kim MY. Epigenetics: an emerging player in gastric cancer. W J Gastroenterol 2014;20(21): 6433-47

9.

Kanwal R, Gupta S. Epigenetic modifications in cancer. Clin Genet 2012; 81(4):303-11

10.

Korkmaz A, Manchester LC, Topal T, et al. Epigenetic mechanisms in human physiology and disease. J Exp Integr Med 2011;1(3):139-47

11.

Inbar-Feigenberg M, Choufani S, Butcher DT, et al. Basic concepts of epigenetics. Fertil Steril 2013;99(3):607-15

12.

Park YS, Jin MY, Kim YJ, et al. The global histone modification pattern correlates with cancer recurrence and overall survival in gastric adenocarcinoma. Ann Surg Oncol 2008;15(7):1968-76

13.

Leszinski G, Gezer U, Siegele B, et al. Relevance of histone marks H3K9me3 and H4K20me3 in cancer. Anticancer Res 2012; 32(5):2199-205

14.

Zhou Q, Chaerkady R, Shaw PG, et al. Screening for therapeutic targets of vorinostat by SILAC-based proteomic

209

Review


analysis in human breast cancer cells. Proteomics 2010;10(5):1029-39


15.

Gos M. Epigenetic mechanisms of gene expression regulation in neurological diseases. Acta Neurobiol Exp (Warsz) 2013; 73(1):19-37

16.

Lin Y, Zhou BP. Histone mimics: digging down under. Front Biol 2013;8(2):228-33

17.

Salam MT, Zhang Y, Begum K. Epigenetics and childhood asthma: current evidence and future research directions. Epigenomics 2012;4(4):415-29

18.

19.

20.

21.

Urdinguio RG, Sanchez-Mut JV, Esteller M. Epigenetic mechanisms in neurological diseases: genes, syndromes, and therapies. Lancet Neurol 2009;8(11):1056-72 Villeneuve LM, Natarajan R. The role of epigenetics in the pathology of diabetic complications. Am J Physiol Renal Physiol 2010;299(1):F14-25

28.

..

An excellent review on MS-based approaches for histone post-translational modifications identification to data analysis.

43.

Soldi M, Cuomo A, Bonaldi T. Improved bottom-up strategy to efficiently separate hypermodified histone peptides through ultra-HPLC separation on a bench top Orbitrap instrument. Proteomics 2014;14: 2212-25

31.

Bhargava P. Epigenetics to proteomics: from yeast to brain. In: Proteomics 2010;10(4): 749-70

32.

Goncalves A, Bertucci F. Clinical application of proteomics in breast cancer: state of the art and perspectives. Med Princ Prac 2011;20:4-18

44.

Galva˜o ERCGN, Martins LMS, Ibiapina JO, et al. Breast cancer proteomics: a review for clinicians. J Cancer Res Clin Oncol 2011;137:915-25

Arnaudo AM, Garcia BA. Proteomic characterization of novel histone post-translational modifications. Epigenetics Chromatin 2013;6:24

45.

Camp RL, Neumeister V, Rimm DL. A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers. J Clin Oncol 2008;26:5630-7

Simo-Riudalbas L, Esteller M. Targeting the histone orthography of cancer: drugs for writers, erasers and readers. Br J Pharmacol 2014. [Epub ahead of print]

46.

Simo-Riudalbas L, Esteller M. Cancer genomics identifies disrupted epigenetic genes. Hum Genet 2014;133(6):713-25

33.

35.

van Zwieten A. Tissue microarray technology and findings for diagnostic immunohistochemistry. Pathology 2013;45: 71-9

47.

Chung L, Baxter RC. Breast cancer biomarkers: proteomic discovery and translation to clinically relevant assays. Expert Rev Proteomics 2012;9(6):599-614

36.

Byrum SD, Raman A, Taverna SD, Tackett AJ. ChAP-MS: a method for identification of proteins and histone posttranslational modifications at a single genomic locus. Cell Reports 2012;2: 198-205

48.

Kocevar N, Hudler P, Komel R. The progress of proteomic approaches in searching for cancer biomarkers. New Biotechnol 2013;30(3):319-26

49.

Bellizzi AM, Frankel WL. Colorectal cancer due to deficiency in DNA mismatch repair function: a review. Adv Anat Pathol 2009; 16(6):405-17

50.

Hudler P. Genetic testing, an optimal strategy for lynch syndrome identification. In: Vogelsang M, editor. DNA alterations in lynch syndrome. Springer; Netherlands: 2013. p. 63-83

51.

Sengupta N, Gill KA, MacFie TS, et al. Management of colorectal cancer: a role for genetics in prevention and treatment? Pathol Res Pract 2008;204(7):469-77

52.

Weller M, Stupp R, Reifenberger G, et al. MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurology 2010;6(1):39-51

53.

Dasilva N, Diez P, Matarraz S, et al. Biomarker discovery by novel sensors based on nanoproteomics approaches. Sensors (Basel) 2012;12(2):2284-308

54.

Gonzalez-Gonzalez M, Jara-Acevedo R, Matarraz S, et al. Nanotechniques in proteomics: protein microarrays and novel

Cordero P, Campion J, Milagro FI, Martinez JA. Transcriptomic and epigenetic changes in early liver steatosis associated to obesity: effect of dietary methyl donor supplementation. Mol Genet Metab 2013; 110(3):388-95

27.

Hon GC, Hawkins RD, Caballero OL, et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res 2012;22(2):246-58

Martinez JA, Milagro FI, Claycombe KJ, Schalinske KL. Epigenetics in adipose tissue, obesity, weight loss, and diabetes. Adv Nutr 2014;5(1):71-81

23.

26.

30.

tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones. Proteomics 2014;14:2200-11

34.

Esteller M. Epigenetics in cancer. N Engl J Med 2008;358(11):1148-59

25.

Choi JY, James SR, Link PA, et al. Association between global DNA hypomethylation in leukocytes and risk of breast cancer. Carcinogenesis 2009;30(11): 1889-97

Berger SL, Kouzarides T, Shiekhattar R, Shilatifard A. An operational definition of epigenetics. Genes Dev 2009;23(7):781-3

22.

24.

29.

Mazzio EA, Soliman KF. Basic concepts of epigenetics: impact of environmental signals on gene expression. Epigenetics 2012;7(2): 119-30 Milagro FI, Mansego ML, De Miguel C, Martinez JA. Dietary factors, epigenetic modifications and obesity outcomes: progresses and perspectives. Mol Aspects Med 2013;34(4):782-812

37.

38.

Yuan Z-F, Arnaudo AM, Garcia BA. Mass spectrometric analysis of histone proteoforms. Annu Rev Anal Chem Palo Alto, Calif 2014;7:113-28

39.

Kellie JF, Tran JC, Lee JE, et al. The emerging process of Top Down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput. Mol Biosyst 2011;6:1532-9

Li C, Zhao S, Zhang N, et al. Differences of DNA methylation profiles between monozygotic twins’ blood samples. Mol Biol Rep 2013;40(9):5275-80 Niederhoffer KY, Penaherrera M, Pugash D, et al. Beckwith-Wiedemann syndrome in sibs discordant for IC2 methylation. Am J Med Genet A 2012;158A(7):1662-9 Toiyama Y, Okugawa Y, Goel A. DNA methylation and microRNA biomarkers for noninvasive detection of gastric and colorectal cancer. Biochem Biophys Res Commun 2014; 455(1-2):43-57

210

Evertts AG, Zee BM, Garcia BA. Modern approaches for investigating epigenetic signaling pathways. J Appl Physiol 1985 2010;109:927-33

40.

41.

42.

Siuti N, Kelleher NL. Decoding protein modifications using top-down mass spectrometry. Nat Methods 2007;4:817-21 Wu S, Lourette NM, Toli N, et al. An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications. 2010;8:1347-57 Sidoli S, Schwa¨mmle V, Ruminowicz C, et al. Middle-down hybrid chromatography/



detection platforms. Eur J Pharm Sci 2012; 45(4):499-506


55.

Mischak H, Coon JJ, Novak J, et al. Capillary electrophoresis-mass spectrometry as a powerful tool in biomarker discovery and clinical diagnosis: an update of recent developments. Mass Spectrom Rev 2009; 28(5):703-24

56.

Hembrough T, Thyparambil S, Liao WL, et al. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue. Clin Proteomics 2012;9(1):5

57.

Fung ET. A recipe for proteomics diagnostic test development: the OVA1 test, from biomarker discovery to FDA clearance. Clin Chem 2010;56(2):327-9

..

An excellent representation of combining the basic research and novel proteomic analytical tools leading towards successful clinical implementation.

58.

59.

60.

.

61.

Bristow RE, Smith A, Zhang Z, et al. Ovarian malignancy risk stratification of the adnexal mass using a multivariate index assay. Gynecol Oncol 2013;128(2):252-9 Goodrich ST, Bristow RE, Santoso JT, et al. The effect of ovarian imaging on the clinical interpretation of a multivariate index assay. Am J Obstet Gynecol 2014;211(1):65 e1- e11 Ramos-Vara JA, Miller MA. When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry–the red, brown, and blue technique. Vet Pathol 2014;51(1): 42-87 Review of all aspects of immunohistochemistry addressing advantages and disadvantages offering key solutions to most common technical issues. Bock I, Dhayalan A, Kudithipudi S, et al. Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays. Epigenetics 2011;6(2): 256-63

62.

Egelhofer TA, Minoda A, Klugman S, et al. An assessment of histone-modification antibody quality. Nat Struct Mol Biol 2011; 18(1):91-3

63.

Hattori T, Taft JM, Swist KM, et al. Recombinant antibodies to histone post-translational modifications. Nat Methods 2013;10(10):992-5

64.

Peach SE, Rudomin EL, Udeshi ND, et al. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications


reveals patterns of co-occurring marks on histone protein molecules. Mol Cell Proteomics 2012;11(5):128-37

Review

cancer patients treated with surgery. Eur J Surg Oncol 2012;38:1051-7 75.

Kumagai T, Akagi T, Desmond JC, et al. Epigenetic regulation and molecular characterization of C/EBPalpha in pancreatic cancer cells. Int J Cancer 2009; 124(4):827-33

Sandoval J, Peiro-Chova L, Pallardo FV, Garcia-Gimenez JL. Epigenetic biomarkers in laboratory diagnostics: emerging approaches and opportunities. Expert Rev Mol Diagn 2013;13(5):457-71

76.

Chang L, Guo F, Wang Y, et al. MicroRNA-200c regulates the sensitivity of chemotherapy of gastric cancer SGC7901/ DDP cells by directly targeting RhoE. Pathol Oncol Res 2014;20(1):93-8

66.

Healey MA, Hu R, Beck AH, et al. Association of H3K9me3 and H3K27me3 repressive histone marks with breast cancer subtypes in the nurses’ health study. Breast Cancer Res Treat 2014;147: 639-51

77.

Lin XC, Zhu Y, Chen WB, et al. Integrated analysis of long non-coding RNAs and mRNA expression profiles reveals the potential role of lncRNAs in gastric cancer pathogenesis. Int J Oncol 2014;45(2): 619-28

67.

Holm K, Grabau D, Lo¨vgren K, et al. Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes. Mol Oncol 2012;6:494-506

78.

Wu HH, Lin WC, Tsai KW. Advances in molecular biomarkers for gastric cancer: miRNAs as emerging novel cancer markers. Expert Rev Mol Med 2014;16:e1

68.

Elsheikh SE, Green AR, Rakha EA, et al. Global histone modifications in breast cancer correlate with tumor phenotypes, prognostic factors, and patient outcome. Cancer Res 2009;69:3802-9

79.

Hudler P, Vogelsang M, Komel R. Genetic instability in gastric cancer. In: Lofty M, editor. Gastric carcinoma - molecular aspects and current advances. InTech; Rijeka: 2010. p. 345

69.

Leroy G, Dimaggio PA, Chan EY, et al. A quantitative atlas of histone modification signatures from human cancer cells. Epigenetics Chromatin 2013;6:20

80.

David S, Meltzer SJ. Stomach - Genetic and epigenetic alterations of preneoplastic and neoplastic lesions. Cancer Biomark 2010; 9(1-6):493-507

70.

Fukushige S, Horii A. Road to early detection of pancreatic cancer: attempts to utilize epigenetic biomarkers. Cancer Lett 2014;342:231-7

81.

.

Review on recent advances of epigenetic biomarkers for timely diagnosis of pancreatic cancer.

Gao F, Ji G, Gao Z, et al. Direct ChIP-bisulfite sequencing reveals a role of H3K27me3 mediating aberrant hypermethylation of promoter CpG islands in cancer cells. Genomics 2014;103(2-3): 204-10

82.

Ding SZ, Fischer W, Kaparakis-Liaskos M, et al. Helicobacter pylori-induced histone modification, associated gene expression in gastric epithelial cells, and its implication in pathogenesis. PLoS One 2010;5(4):e9875

83.

Zhang L, Zhong K, Dai Y, Zhou H. Genome-wide analysis of histone H3 lysine 27 trimethylation by ChIP-chip in gastric cancer patients. J Gastroenterol 2009;44(4): 305-12

84.

Lao VV, Grady WM. Epigenetics and colorectal cancer. Nat Rev Gastroenterol Hepatol 2011;8(12):686-700

85.

Cheah PY. Recent advances in colorectal cancer genetics and diagnostics. Crit Rev Oncol Hematol 2009;69(1):45-55

86.

Bogaert J, Prenen H. Molecular genetics of colorectal cancer. Ann Gastroenterol 2014; 27(1):9-14

87.

Enroth S, Rada-Iglesisas A, Andersson R, et al. Cancer associated epigenetic

.

A series of histone modifications showing important correlation with different tumor grades, morphologic types and phenotype subclasses of breast cancer.

65.

71.

72.

73.

74.

Hinton J, Callan R, Bodine C, et al. Potential epigenetic biomarkers for the diagnosis and prognosis of pancreatic ductal adenocarcinomas. Expert Rev Mol Diagn 2013;13:431-43 Manuyakorn A, Paulus R, Farrell J, et al. Cellular histone modification patterns predict prognosis and treatment response in resectable pancreatic adenocarcinoma: results from RTOG 9704. J Clin Oncol 2010;28: 1358-65 Wei Y, Xia W, Zhang Z, et al. Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers. Mol Carcinog 2008;47:701-6 Watanabe T, Morinaga S, Akaike M, et al. The cellular level of histone H3 lysine 4 dimethylation correlates with response to adjuvant gemcitabine in Japanese pancreatic

211

Review


of human colon cancer cells in a p53- and DNMT1(DNA methyltransferase 1)independent manner. Biochem J 2013; 449(2):459-68

transitions identified by genome-wide histone methylation binding profiles in human colorectal cancer samples and paired normal mucosa. BMC Cancer 2011;11:450 88.


89.

90.

Brinkman AB, Gu H, Bartels SJ, et al. Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res 2012;22(6):1128-38

91.

Benard A, Goossens-Beumer IJ, van Hoesel AQ, et al. Nuclear expression of histone deacetylases and their histone modifications predicts clinical outcome in colorectal cancer. Histopathology 2015; 66(2):270-82

92.

Jin L, Hanigan CL, Wu Y, et al. Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression

212

93.

Guo C, Chen LH, Huang Y, et al. KMT2D maintains neoplastic cell proliferation and global histone H3 lysine 4 monomethylation. Oncotarget 2013; 4(11):2144-53 Hashimoto T, Yamakawa M, Kimura S, et al. Expression of acetylated and dimethylated histone H3 in colorectal cancer. Dig Surg 2013;30(3):249-58 Nakazawa T, Kondo T, Ma D, et al. Global histone modification of histone H3 in colorectal cancer and its precursor lesions. Hum Pathol 2012;43(6):834-42

94.

Benard A, Goossens-Beumer IJ, van Hoesel AQ, et al. Histone trimethylation at H3K4, H3K9 and H4K20 correlates with patient survival and tumor recurrence in early-stage colon cancer. BMC Cancer 2014;14:531

95.

Yokoyama Y, Hieda M, Nishioka Y, et al. Cancer-associated upregulation of histone H3 lysine 9 trimethylation promotes cell motility in vitro and drives tumor formation in vivo. Cancer Sci 2013;104(7):889-95

96.

Karczmarski J, Rubel T, Paziewska A, et al. Histone H3 lysine 27 acetylation is altered in colon cancer. Clin Proteomics 2014; 11(1):24


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