A STUDY OF TUMOR SUPPRESSOR GENE EXPRESSION AND PROMOTER METHYLATION FOR THE IDENTIFICATION OF PROGNOSTIC MARKERS IN GLIOBLASTOMA

(1)

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Paulina Vaitkienė

A STUDY OF TUMOR SUPPRESSOR GENE

EXPRESSION AND PROMOTER

METHYLATION FOR THE IDENTIFICATION

OF PROGNOSTIC MARKERS IN

GLIOBLASTOMA

Doctoral Dissertation Biomedical Sciences, Biology (01B) Kaunas, 2013

(2)

2

Dissertation has been prepared at the Medical Academy of Lithuanian University of Health Sciences during the period of 2005–2013.

Scientific Supervisor

Prof. Dr. Dainius H. Pauža (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Biology – 01B)

Consultant

Prof. Dr. Habil. Arimantas Tamašauskas (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Medicine – 06B)

(3)

3

LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Paulina Vaitkienė

GLIOBLASTOMŲ PROGNOZINIŲ

ŽYMENŲ PAIEŠKA TIRIANT NAVIKĄ

SLOPINANČIŲ GENŲ RAIŠKĄ BEI

PROMOTORIŲ METILINIMĄ

Daktaro disertacija Biomedicinos mokslai,

biologija (01B)

(4)

4

Disertacija rengta 2005–2013 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijoje.

Mokslinis vadovas

prof. dr. Dainius H. Pauža (Lietuvos sveikatos mokslų universitetas, Medicinos akademija, biomedicinos mokslai, biologija – 01B)

Konsultantas

prof. habil. dr. Arimantas Tamašauskas (Lietuvos sveikatos mokslų universitetas, Medicinos akademija, biomedicinos mokslai, medicina – 06B)

(5)

5

6 5.5.1. AREG methylation ... 74 5.5.2. CASP8 methylation ... 76 5.5.3. CD81 methylation ... 78 5.5.4. DcR1 methylation ... 80 5.5.5. DR4 methylation ... 82 5.5.6. GATA4 methylation ... 84 5.5.7. GATA6 methylation ... 86 5.5.8. hMLH1 methylation ... 88 5.5.9. NTPX2 methylation ... 89 5.5.10. TES methylation ... 91 5.5.11. TFPI2 methylation ... 93

5.6. Gene comethylation and its associations with survival ... 96

DISCUSSION ... 104 CONCLUSIONS ... 113 REFERENCES ... 114 LIST OF PUBLICATIONS ... 129 ACKNOWLEDGMENTS ... 134 APPENDIX ... 135

(7)

7

ABBREVIATIONS

AREG amphiregulin

CASP8 caspase 8

CD81 CD81 molecule, TAPA-1

CNS central nervous system

COX7A1 cytochrome c oxidase subunit VIIa polypeptide 1

CpG "p" phosphodiester bond between the cytosine and guanine nucleotides.

CSC cancer stem cells

DcR1 tumor necrosis factor receptor superfamily,

member 10c

DNA deoxyribonucleic acid

DNMT DNA methyltransferases

DR4 tumor necrosis factor receptor superfamily, member 10a

ECM extracellular matrix

EGFR epidermal growth factor receptor

GBM glioblastoma

GATA4 GATA binding protein 4

GATA6 GATA binding protein 6

G-CIMP glioma- CpG island methylator phenotype

H3K4 lysine 4 on histone H3

HIF-1 hypoxic inducible factor 1

hMLH1 MutL homolog 1

IDH isocitrate dehydrogenase enzyme

KRT81 keratin 81

MBD DNA methyl-binding domain

MeCP2 methyl CpG-binding proteins

MGMT O6-methylguanine DNA methyltransferase

MMP-2 matrix metalloproteinase 2

MMR DNA mismatch repair system

MSP methylation-specific polymerase chain reaction

(8)

8

PCR polymerase chain reaction

PDGF platelet-derived growth factor

RT-PCR reverse-transcription polymerase chain reaction

SPINT1 serine peptidase inhibitor

TES testin

TFPI-2 tissue factor pathway inhibitor 2

TRAIL tumor necrosis factor-related apoptosis-inducing ligand

VEGF vascular endothelial growth factor

VEGFR vascular endothelial growth factor receptor

WHO World Health Organization

5hmC 5-hydroxymethylcytosine

(9)

9

INTRODUCTION

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Patients with glioblastoma have a median survival time of only 12-15 months (1, 2), and prognosis remains extremely poor despite multimodal treatment by surgery, radiotherapy and, chemotherapy. Generally, prognostically favorable clinical factors, such as young age and a good Karnofsky performance score at diagnosis are associated with a better prognosis. However, various clinical characteristics fail to explain a variation in disease progression and response to treatment in histologically diagnosed glioblastomas. There is increasing interest in identifying the molecular markers that better capture the status of a tumor in order to improve the existing predictions (3). The identification of methylated genes in cancer may provide an insight in the molecular mechanisms of tumor development and might reveal new tools to define the markers of prognostic significance (4). For example, the epigenetic silencing of the tumor suppressor gene O6-methylguanine DNA methyltransferase (MGMT), which encodes a DNA repair enzyme, sensitizes cancer cells to alkylating agents and is associated with a significantly improved outcome in GBM patients treated with radiotherapy and temozolomide (5).

Recently, several microarray-based GBM studies have identified the gene CpG rich promoter-associated sequences that are frequently methylated. When hypermethylation occurs within the promoter region of a gene, it could inhibit its expression, contributing to the inactivation of tumor suppressor genes in cancer and provide the cell with a growth advantage (6). The list of genes reported as being silenced in gliomas by aberrant promoter methylation is growing and includes targets involved in glioma development and progression. Up to now, a lot of genes were identified to be differentially methylated in a subset of gliomas, enabling the discrimination of tumors with different biological and clinical properties according to the methylation pattern (2, 7, 8). However, studies are usually limited because of a small sample of glioblastomas and scarce clinical data.

In order to identify the molecular markers relevant to the development, diagnosis, and prognosis of glioblastoma, the analysis of 14 genes (AREG, CASP8, CD81, COX7A1, DcR1, DR4, GATA4, GATA6, hMLH1, KRT81, NPTX2, SPINT1, TES, and TFPI-2) was performed. The products of these genes inhibit cellular migration and metastasis, regulate cell proliferation, and protect the genome from mutagenesis. The main objective of our study was to further characterize the role of the latter genes in the pathogenesis of glioblastoma and to evaluate its clinical importance.

(10)

10

1. AIM AND OBJECTIVES OF THE STUDY

The aim of this study was to determine aberrant promoter methylation of tumor suppressor genes in tumor tissue DNA from patients with glioblastoma multiforme and to evaluate the associations between the methylation profile of genes, patients’ clinical characteristics, and prognostic value.

The objectives of the study were as follows:

1. To investigate the expression of 5 genes (AREG, COX7A1, KRT81, NPTX2, and SPINT1) in 22 different glioblastoma samples and to compare with the expression of these genes in control brain tissue samples;

2. To determine if the differences in the expression of AREG, COX7A1, KRT81, NPTX2, and SPINT1 genes are associated with the promoter methylation of these genes;

3. To determine the methylation frequency of 11 genes (AREG, CASP8, CD81, DcR1, DR4, GATA4, GATA6, hMLH1, NPTX2, TES, and TFPI2), involved in cell motility, growth, adhesion, apoptosis, and repair processes, in 100 glioblastoma tissue samples;

4. To evaluate the associations between AREG, CASP8, CD81, DcR1, DR4, GATA4, GATA6, hMLH1, NPTX2, TES, and TFPI2 gene promoter methylation and clinical characteristics of patients with glioblastoma.

5. To determine the relationship between AREG, CASP8, CD81, DcR1, DR4, GATA4, GATA6, hMLH1, NPTX2, TES, and TFPI2 gene promoter methylation and patients’ survival after surgery;

6. To identify epigenetic markers and their combinations that could be significant in the prognosis of patients with glioblastoma.

(11)

11

2. ORIGINALITY OF THE STUDY

Glioblastoma is one of the most aggressive tumors with a poor prognosis. The epigenetic events that regulate gene expression have clearly emerged as a fundamental mechanism in developmental biology and in the pathogenesis of cancer. For example, multiple genes that affect numerous cellular pathways are silenced by hypermethylation in cancer, and studies of these genes have increased our understanding of how cancer develops and progresses.

In 2007, Mueller et al. succeeded in unveiling novel candidate genes, which are epigenetically regulated in glioblastomas, by using the pharmacologic manipulation of glioma cells with the demethylating agent 5´-aza-dC combined with genome wide expression profiling (7). To confirm the importance of these genes in gliomagenesis, in this study, a more detailed analysis of COX7A1, SPINT1, AREG, NPTX2, and KRT81 gene expression and their promoter methylation in glioblastoma samples was performed for the first time. Genes were evaluated by using the following methods: reverse-transcription polymerase chain reaction (RT-PCR), bisulfite sequencing, and methylation-specific PCR (MSP). Potential epigenetic markers in glioblastoma – AREG and NPTX2 – the expression of which was found to be associated with promoter methylation have been identified.

Moreover, in this study, the methylation frequency of 11 genes (AREG, CASP8, CD81, DcR1, DR4, GATA4, GATA6, hMLH1, NPTX2, TES, and TFPI2), involved in cell motility, growth, adhesion, apoptosis, and repair processes, was determined in 100 glioblastoma tissue samples. The data on the frequency of methylation of these genes in glioblastoma are scarce to evaluate the usability and suitability of new epigenetic markers in molecular diagnosis of these tumors and prognosis of the disease.

To our knowledge, the methylation frequencies of the AREG, DcR1, GATA4, NPTX2, and TFPI2 gene promoters in glioblastoma have not been reported in literature; therefore, we believe that our study was the first to determine the methylation frequencies and significantly contributed to the existing knowledge on epigenetic markers in glioblastoma and the process of gliomagenesis.

Our study differs from other published studies by a sample size, analysis of clinical data, and identification of new methylation markers and their combinations that could be used in the diagnosis and prognosis of glioblastoma.

(12)

12

To our knowledge, this study is the first to report the significant associations between the methylation of the AREG, CASP8, GATA6, and TFPI2 gene promoters and patients’ survival after surgery. After a detailed investigation of these genes and confirmation of the data in larger scale studies, they could be used for the molecular typing of glioblastomas and more accurate diagnosis to tailor the treatment strategies and prognosticate the course of the disease.

Moreover, this study is one of the first studies where not only the impact of the methylation of single gene promoters on survival has been studied, but also associations between the comethylation of several genes and patients’ clinical characteristics have been determined. With the help of comprehensive statistical analysis, a unique set of 6 genes (AREG, CASP8, DR4, GATA4, GATA6, and TFPI2) has been identified; the methylation studies of the promoters of this gene combination would help reliably predict survival as compared with the analysis of individual genes.

The importance of this combination of genes in the diagnosis and prognosis of disease course in glioblastoma needs to be evaluated and validated in more detailed studies with a larger cohort of patients with glioblastoma.

In this study, the fundamental investigations were linked to clinical data. The study conclusions are undoubtedly useful for clinical practice, and the obtained results provide additional information for the future planning of new clinical trials.

(13)

13

3. REVIEW OF LITERATURE

3.1. Glioblastoma epidemiology and morphology

Central nervous system (CNS) tumors in Europe have an incidence of approximately 6-7 per 100 000 persons per year and account for 2% of all cancer-related deaths (9). The diagnosis and grading of primary brain tumors follows the World Health Organization (WHO) classification (10). The WHO classification indicates malignant potential, response to treatment, and survival. Gliomas are tumors that are thought to arise from glial progenitor and glial cells. These tumors account for 32% of all primary brain tumors and, and 80% of all malignant primary brain tumors diagnosed in the United States (11). The overall annual incidence of gliomas varies from 5 to 6 cases per 100 000 worldwide (12). Glial tumors are categorized into 4 grades. Grade I and II low-grade astrocytomas are slow-growing, less aggressive tumors, while grade III and IV high-grade gliomas are malignant tumors, characterized by a high proliferation rate (grade III) and the presence of necrotic tissue and/or angiogenic activity (grade IV) (13). Glioblastoma is also known as grade IV astrocytoma or glioblastoma multiforme. The term multiforme refers to the vast intratumoral heterogeneity seen in the disease. The histopathology of glioblastoma shows brisk mitotic activity, microvascular proliferations, thrombosis, and necrosis (14). Glioblastoma multiforme accounts for up to 60% of all malignant primary brain tumors in adults, occurring in 2-3 cases per 100 000 in Europe and North America (15). It is one of the most devastating and lethal forms of human cancer. GBM presents some of the greatest challenges in the management of cancer patients worldwide, despite notable recent achievements in oncology. Even with aggressive surgical resections using state-of-the-art preoperative and intraoperative neuroimaging, along with recent advances in radiotherapy and chemotherapy, the prognosis for GBM patients remains dismal: survival time after diagnosis is about 1 year (16). Only 3%–5% of patients survive longer than 5 years (17). Nevertheless, residual tumor causes relapse in almost all cases. GBM most frequently recurs after a median survival time of 32 to 36 weeks (18). Although GBM can manifest itself at any age, it preferentially occurs in adults, with a peak age of incidence between 45 and 70 years (19). This type of cancer is more common in men than women. It most frequently involves the brain hemispheres, but it can also affect basal ganglia and the brain stem (19). The etiology of malignant glioma is unknown. Therapeutic ionizing irradiation has been confirmed as a sole unequivocal risk factor for the development of

(14)

14

glioblastoma. No association between exposure to electromagnetic fields and development of brain tumors has been established so far. Further, no definite association between viral infection or diet (e.g., intake of N-nitroso compounds) and malignant glioma has been proven as well (14).

Invasion

An additional important feature of malignant glioma include its great tendency to infiltrate into normal brain tissue, rendering the condition incurable by surgery alone (19). Migrating glioma cells are relatively resistant to cytotoxic therapy (chemotherapy, irradiation) because the processes of metabolism are reduced as compared to nonmigrating tumor cells. The invasion of glioma cells requires interaction with the extracellular matrix (ECM) and with surrounding cells of the healthy brain tissue. The brain ECM is mainly composed of soft components such as glycosaminoglycans, proteoglycans, tenascin-C, and thrombospondin (14). Rigid components like collagen, fibronectin, or laminin are limited to perivascular and vascular areas in the brain. Migrating glioma cells may degrade the soft components of ECM by proteases, namely the matrix metalloproteinases, serine proteases, and cysteine proteases (14). The outer GBM border usually observed in T1-weighted gadolinium magnetic resonance imaging does not delineate the true dimension of the tumor. Moreover, tumor cells diffusely invade the normal brain and may be detected beyond that border (19).

Hypoxia and angiogenesis

Vascular proliferation and tissue necrosis are characteristic features of malignant gliomas, particularly of glioblastoma. These features are most likely the consequence of rapidly increasing tumor mass that is inadequately oxygenized by the preexisting vasculature (14). Hypoxia-related genes are significantly associated with necrosis in human glioblastoma specimens, which suggests that there is a possible link between glioblastoma necrosis and hypoxia. Hypoxic cells are known to overexpress hypoxic inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), and matrix metalloproteinase 2 (MMP-2) (20). HIF-1 is translocated to the nucleus and induces the transcription of target genes involved in angiogenesis, migration, proliferation, and cell survival. The downstream effectors of HIF-1 include VEGF and vascular endothelial growth factor receptor (VEGFR). VEGF is a major angiogenic growth factor. It binds to and activates the tyrosine kinase receptors of the VEGFR family. Activated

(15)

15

tyrosine kinase activates downstream pathways that induce proliferation and migration of vascular cells (14). Considering the morphology of glioblastoma necrosis, these factors could cause tumor cell migration to form pseudopalisades and microvascular hyperplasia in perinecrotic areas, which are associated with poor prognosis in cancer (20). Tumor cells up-regulate hypoxia-associated factors that induce angiogenesis. In malignant gliomas, newly formed vessels do not show a normal phenotype, but appear as bizarre vascular proliferates. It is assumed that these bizarre vascular proliferates are not suitable to sufficiently oxygenize the tumor tissue, resulting in a vicious circle of hypoxia and aberrant angiogenesis (14).

Glioblastoma cancer stem cell hypothesis

Traditionally, it was thought that the adult human brain does not contain precursor cells, and it was assumed that brain tumors derive from mature parenchymal cells (14). An increasing body of evidence has strongly supported the concept that a subpopulation of cancer cells in each tumor has a greater potential of cancer initiation and repopulation. These cells are known as cancer stem cells (CSCs) or tumor-initiating or propagating cells because they share some critical characteristics with normal stem cells (21). CSCs are defined as tumor cells with stem cell properties, i.e., asymmetric cell division, infinite growth, and multipotency, that later differentiate into rapidly proliferating progenitor-like and more differentiated cells, defining the histopathology of the tumor (22). The CSC hypothesis also suggests that resistance to chemo- and radiotherapy is driven by CSCs and thus challenges the present diagnostic and therapeutic strategies (22). It has been suggested that cancer stem cells are the main proliferating cell population in malignant gliomas responsible for tumor growth and progression (14). GBM stem cells may be resulted from genetic and epigenetic changes in neural stem/progenitor cells or the differentiated cells such as astrocytes after a series of mutations or epigenetic reprogram. Although the origin of GBM stem cells is not clearly defined, they share similar properties with normal neural stem cells that endow these cells with key traits in carcinogenesis (21).

Pathogenesis of glioblastomas

Gliomas, like all human tumors, arise by a step-wise accumulation of genetic events that inactivate tumor suppressor genes and activate oncogenes (23). Glioblastoma may develop from diffuse low-grade or anaplastic astrocytomas (secondary glioblastoma), but more frequently, they

(16)

16

manifest de novo, without a less malignant precursor lesion (primary glioblastoma) (14). Primary and secondary GBMs are histologically indistinguishable, but at the molecular level, each type demonstrates distinct patterns of genetic alterations and these have long been thought to represent different routes of molecular pathogenesis to a common histopathologic end point (24). Two genetic alterations have been detected to occur at a significant rate in primary GBM: amplification or activating mutation of epidermal growth factor receptor (EGFR) and loss of chromosome 10 (23). The activation of growth factor receptors – EGFR, alpha-type platelet-derived growth factor receptor, or MET – by mutation and amplification inhibits the functions of tumor suppressors such as p53 and Rb (24). The tumor suppressor gene PTEN (phosphatase and tensin homolog) is mutated in the 10q23 region in approximately one-third of primary GBMs (19). Mutations in this gene have been described only in malignant gliomas and are rarely associated with p53 mutations (19). Other frequent mutations in primary GBM affect the CDK cell-cycle-regulator genes CDK4/6 and CDKN2A/CDKN2B (19). In summary, primary glioblastoma typically arises with a complement of mutations that, one way or another, serves to activate progrowth pathways and inactivate or suppress tumor suppressors p53 and Rb. Because primary GBMs arise de novo, there is little information on the sequence of events during pathogenesis (24).

Other GBMs arise by the step-wise progression through a series of lower grade lesions (low-grade astrocytoma, anaplastic astrocytoma) and are called secondary GBMs (23). A number of alteration have been noted in lower grade lesions, including the loss of the tumor suppressors p53, p15, p16, and Rb and overexpression of the platelet-derived growth factor (PDGF) (23). Somatic mutations of the isocitrate dehydrogenase enzymes (IDH1 and IDH2) appears to play a critical and early role of these gliomas (24). The recent finding of mutations in IDH genes in 70%-80% of grade II and III astrocytomas, as well as some glioblastomas, has provided a unifying mechanism for early gliomagenesis. As IDH enzymes are typically associated with the Krebs cycle, they play a multitude of other roles in normal cellular biology, such as contributing to cellular defenses against oxidative damage and, potentially, regulating epigenetic modiﬁers (25). The incidence of IDH mutation in primary GBM is low (~5%), reinforcing the notation of a distinct pathogenesis associated with tumors arising in this clinical scenario (24).

Despite the genetic differences, no differences in sensitivity to conventional chemotherapy between primary and secondary glioblastoma multiforme have been reported. The molecular and genetic aberrations in

(17)

17

There are not many molecular factors that contribute to long-term survival. Among the molecular markers, TP53 mutation, infrequent amplification of the EGFR gene, combined LOH 1p/19q, loss of chromosome arm 19q, and low rate of tumor proliferation have been associated with long-term survival in GBM (26, 27). MGMT promoter methylation is seen in ~50% of glioblastomas and has been linked to prolonged progression-free and overall survival (1). PTEN protein expression was a favorable factor for long-term survival (27). In addition, mutations in the IDH1 and IDH2 genes have recently been reported in a subset of glioblastomas from younger patients and linked to a more favorable survival outcome (29). So far, the molecular and cellular mechanisms of long-term survival in glioblastoma patients are not known.

3.2. The concept of DNA methylation

Glioblastomas arise by a step-wise accumulation of the events that inactivate tumor suppressor genes and activate oncogenes. These can occur by both epigenetic regulatory mechanisms and structural genomic alterations such as overexpression and amplification activating oncogenes, and transcriptional silencing and genomic deletion inactivating tumor suppressor genes (23).

Epigenetics has emerged as an important field in biomedical research in the last decade, describing the novel mechanisms of normal biological processes as well as disease pathogenesis. Epigenetics can be defined as the study of heritable changes of a phenotype such as the gene expression patterns of a specific cell type that are not caused by changes in the nucleotide sequence of genetic code itself (30).

Epigenetic regulatory mechanisms are as follows: 1. Posttranslational histone modification;

2. ATP-dependent chromatin remodeling complexes; 3. Polycomb/trithorax protein complexes;

4. Small and other noncoding RNA (siRNA and miRNA); 5. DNA methylation.

These mechanisms are discrete, but interact closely in order to regulate gene expression.

Many of the changes, particularly those resulting from DNA methylation and histone deacetylation, affect gene expression and genome stability through the inappropriate regulation of a local chromatin structure. Furthermore, recent data suggest that epigenetic changes are involved in the earliest phases of tumorigenesis, and that they may predispose stem (progenitor) cells to subsequent genetic and epigenetic changes that are

(18)

18

involved in tumor promotion. Given the observed frequency of DNA methylation changes in tumorigenesis and the inherent stability of these molecular abnormalities, these events may provide ideal biomarkers for molecular diagnostics and early detection of cancer (19).

DNA methylation is currently the most widely studied form of epigenetic programming. This postreplication modification is almost exclusively found on the 5 position of the pyrimidine ring of cytosines in the context of the dinucleotide sequence CpG (31) (Figure 3.2.1).

Figure 3.2.1. Mechanism of CpG methylation

(A) CpG islands consist of nucleotide bases where cytosine (C) residues are followed by guanine (G) in a genomic DNA sequence. (B) Methylation of CpG islands is catalyzed by DNA methyltransferases (DNMTs), which transfer the methyl group from S-adenosyl-methionine (SAM-CH3) to cytosine. The methylation reaction yields S-adenosyl homocysteine (SAH) and 5-methylcytosine (5mC) (32).

In the term “CpG sites,” “p” represents the phosphodiester bond between the cytosine and guanine nucleotides. The methylation of CpG rich clusters, called CpG islands, is important because they are found in the promoter region and first exons of many genes. CpG islands are regions of ~500 bp to 1 kbp where CpG nucleotides are about 5 times more abundant compared with the rest of the genome (33). Although CpG islands account for approximately 1% of the total human genome, they are present in >50% of

(19)

19

human gene promoters indicating their functional importance in transcriptional control.

DNA methylation is mediated by 1 of 3 DNA methyltransferases (DNMTs), which catalyze the addition of a methyl group from a universal methyl donor, S-adenosyl-L-methionine, to the 5-carbon position of a cytosine (31). DNMT1 is considered to be primarily responsible for the maintenance of this epigenetic modification and copies the pre-existing methylation pattern onto the daughter strand after DNA replication. DNMT3A and DNMT3B are de novo methyltransferases and can also complete the methylation process and correct the errors (31).

CpG islands are variable methylated as a mechanism to regulate gene transcription. They are mostly nonmethylated associated with the maintenance of an open chromatin structure and a potentially active state of transcription (30). Methylation above normal levels is referred to as hypermethylation. The hypermethylation of CpG islands in a gene promoter blocks the ability of transcription factors to interact with the promoter and inhibits gene expression (32) (Figure 3.2.2.).

Several mechanisms have been proposed to be associated with transcriptional repression by DNA methylation. The first mechanism involves direct interference with the binding of specific transcription factors to their recognition sites in their respective promoters. Several transcription factors, including AP-2, c-Myc/Myn, the cyclic AMP-dependent activator CREB, E2F, and NFκB, recognize sequences that contain CpG residues, and binding of each has been shown to be inhibited by methylation (35).

The second mode of repression involves a direct binding of specific transcriptional repressors to methylated DNA (35). In addition to the proteins that create and maintain the methylation pattern, there are proteins that recognize methylated DNA and interact with it. DNA methyl-binding domain (MBD1-4) proteins or methyl CpG-binding proteins (MeCP2) recognize and bind to methylated DNA. They recruit transcriptional corepressors such as histone-deacetylating complexes, polycomb proteins, chromatin-remodeling complexes and attract chromodomain-binding proteins (30).

Methylated DNA can also be bound by some zinc finger proteins such as Kaiso and more recently discovered ZBTB4 and ZBTB38 proteins that are also able to repress transcription in a methylation-dependent manner (30).

(20)

20

Figure 3.2.2. Epigenetic patterns in normal and cancer cells

(A) DNA methylation. In normal cells, nearly all of the CpG dinucleotides are methylated whereas CpG islands, mostly residing in 5′ regulatory regions of genes, are unmethylated. In cancer cells, many CpG islands become hypermethylated, in conjunction with silencing of their cognate genes, while global hypomethylation, mostly at repetitive elements, occurs. (B) Chromatin and histone modification. Active genes are associated with acetylation of histone tails, methylation of lysine 4 on histone H3 (H3K4), and nucleosome depletion at their promoters. The promoters of silenced genes (drawn here in conjunction with DNA hypermethylation) become associated with nucleosomes, lose acetylation and H3K4 methylation marks, and gain repressive methylation marks such as lysine 9 or 27 on histone H3, which recruit repressive complexes. Methylated DNA binding proteins link methylated DNA with the histone modification and nucleosome remodeling machineries (not shown) (34).

DNA methylation can also affect histone modifications and chromatin structure, which, in turn, can alter gene expression. DNA methylation and histone modifications function in a close interplay with nucleosome remodeling and positioning complexes that bind specifically modified histones and methyl CpG-binding proteins, and move nucleosomes on DNA by ATP-dependent mechanisms (34). Nonmethylated CpG island promoters are usually hypersensitive to nucleases and are relatively depleted of nucleosomes, whereas methylated promoters have nucleosomes on them and are nuclease resistant (34).

(21)

21

DNA methylation is possibly a reversible step, and there are indications that under some conditions, the methylated DNA pattern is a balance between active demethylation and remethylation. Demethylation in the absence of DNA replication normally occurs in the zygote and in the development of germ cells, but it also is active in malignant cells. The molecular mechanisms for demethylation remain unclear, though recent studies suggest that DNMT3A and 3B may be involved (31).

In many cases, methylated and silenced genes can be reactivated using DNA methylation inhibitors such as 5-azacytidine. However, it should be noted that an unmethylated state of a CpG island does not necessarily correlate with the transcriptional activity of the gene, but rather that the gene can be potentially activated. On the other hand, the presence of methylation alone does not necessarily induce silencing of nearby genes. Only when a specific core region of the promoter that is often, but not necessarily, spanning the transcription start site becomes hypermethylated, the expression of the associated gene is modified (30).

3.3. DNA methylation in gliomas

In glioblastomas, the changes in methylation patterns can be seen, which are characterized by a genome-wide loss of methylation combined with locus-specific hypermethylation. Hypomethylation may promote tumor growth by the activation of oncogenes, loss of imprinting, or promotion of genomic instability.

CpG island promoter methylation occurs in genes with diverse functions related to tumorigenesis and tumor progression, including cell-cycle regulation, DNA repair, apoptosis, angiogenesis, invasion, and drug resistance. CpG hypermethylation in gliomas may also occur in genes that are not expressed in the brain, suggesting not all events of CpG island methylation are functionally important for tumorigenesis (33).

Several epigenetic alterations have been described in glioblastoma. Among them, the methylation of MGMT has been the most studied. MGMT encodes a DNA repair protein that removes alkyl adducts at the O6 position of a guanine and protects normal cells from carcinogens. Unfortunately, it can also protect tumor cells from chemotherapeutic alkylating agents such as temozolomide. von Deimling et al. in their article summarized that GBM patients with a hypermethylated MGMT promoter demonstrated the survival rates of 49% and 14% at 2 and 5 years, respectively, when treated with concomitant and adjuvant temozolomide and radiotherapy. In contrast, the estimated 2- and 5-year survival rates were only 24% and 5%, respectively,

(22)

22

in similar patients that were initially treated with radiotherapy alone. GBM patients whose tumors lacked MGMT hypermethylation demonstrated the 2- and 5-year survival rates of 15% and 8%, when they received combined radiochemotherapy, and the rates decreased to 2% and 0% if radiotherapy was administered alone (36).

There are data that different-grade gliomas show differences in the methylation status of various genes (37-39). In this context, the methylation of the DNA repair gene MGMT is more frequently observed in secondary GBMs (about 75%) than primary GBMs (36%) or low-grade astrocytomas (48%) (19). Secondary GBMs have a higher overall frequency of promoter methylation compared to primary GBMs at least for the promoters of p14, p16, RB1, MGMT, and TIMP-3. Low-grade gliomas and secondary GBMs show PTEN promoter methylation (33). The progression of glioma over time is associated with distinct epigenetic patterns. Malignant progression and shorter survival in astrocytoma are associated with p14, but not MGMT hypermethylation, suggesting that these are 2 distinct pathways in astrocytoma with different clinical consequences (33). The association between methylation levels and WHO grading may directly suggest that hypermethylation reflects a potential of tumor aggressiveness. On the other hand, there are data that methylation profiles are remarkably stable across glioma evolution, even during anaplastic transformations, suggesting that epigenetic alterations occur early during gliomagenesis (6).

There are some data that the analysis of the concomitant hypermethylation of several genes allows better prediction of survival than that of one gene (9).

Noushmer et al. reported that gliomas could be stratified by the glioma CpG island methylator phenotype (G-CIMP) status into 2 distinct subgroups with different molecular and clinical phenotypes. These molecular classifications have implications for differential therapeutic strategies for glioma patients (8).

Genes involved in invasion and metastasis can also be affected by promoter hypermethylation in gliomas. The CpG promoter hypermethylation of the protocadherin-gamma subfamily is frequently observed in astrocytomas, glioblastomas, and glioma cell lines (33). The significance of CpG island promoter hypermethylation in GBM pathogenesis is emphasized by the observation of epigenetically mediated inactivation of a wide variety of genes associated with tumor suppression (RB1, VHL, EMP3), cell cycle regulation (p16, p15), DNA repair (MGMT, hMLH1), tumor invasion, and apoptosis (19).

(23)

23

3.4. DNA methylation in medical research and practice

With a growing body of knowledge about the pathogenesis of glioblastoma, there is hope for the development of novel therapeutic agents that may effectively treat patients. DNA methylation is potentially preventable or possibly reversible. For example, blocking DNA methylation by inhibiting DNMTs results in the demethylation of CpG islands in daughter cells, with subsequent restored expression of tumor suppressor genes and abrogation of tumor growth (40).

Successful conventional chemotherapy depends on the activation of proapoptotic genes that respond to cytotoxic agents leading to cell death. DNA methylation of these proapoptotic genes can block cell death from occurring leading to chemotherapeutic resistance, thus the reactivation of epigenetically silenced apoptotic genes can increase the efficacy of chemotherapy (41). Many compounds have been discovered to target proteins that control DNA methylation. Some of them are already being used clinically. The most investigated DNA methylation inhibitors are as follows (41):

1. 5-Azacytidine (5-Aza-CR; Vidaza) is a nucleoside analog that incorporates into RNA and DNA and is FDA approved to treat high-risk myelodysplastic syndromes (MDS) patients; successful clinical results have recently been reported.

2. 5-Aza-2 deoxycytidine (5-Aza-CdR; Decitabine) is the deoxy derivative of 5-Aza-CR and is incorporated only into DNA.

3. Zebularine, a cytidine analog that acts similarly to 5-Aza-CR, but has lower toxicity, increased stability and specificity.

4. S110, a decitabine derivative that has increased stability and activity, which has shown promise in preclinical studies.

Two clinically used compounds with DNA demethylating activities – azacitidine (5-azaCR, Vidaza; Celgene, Summit, NJ, USA) and decitabine (5-aza-2′-deoxycytidine, Dacogen; SuperGen, Dublin, CA, USA) – are structurally similar to cytosine nucleosides (42). These inhibitors are incorporated into DNA and trap the DNMT leading to a covalent protein-DNA adduct, depletion of DNMTs, and subsequent demethylation of genomic DNA during replication. Although proven to be potentially effective as antitumor agents in laboratory experiments and in clinical trials, both inhibitors are unstable in solution and can be toxic (43). DNA methylation inhibitors act during S-phase of the cell cycle, thus they preferentially affect rapidly growing cells. In addition to inhibiting DNA methyltransferase activity, azanucleosides also act through unspecific mechanisms, which likely contribute to their clinical effectiveness (41).

(24)

24

Administration of a combination of DNMT and HDAC could have a synergistic effect on gene activation and could allow lower doses of each drug to be used (33).

Zebularine is a proven nucleoside DNMT inhibitor effective at reactivating the silenced genes in vitro and in vivo with a high specificity for cancer cells relative to normal cells. It was demonstrated that Zebularine selectively sensitizes the brain tumor cells that are deficient in DNA-dependent protein kinase. The sensitization of cell killing by Zebularine is mediated by a combination of DNA repair and cell cycle checkpoint defects in DNA-dependent protein kinase-deficient glioblastoma cells (43).

The methylation-induced silencing of cancer-testis antigens, such as NY-ESO-1, can protect cancer cells from being recognized by T cells. Treating cancer cells with demethylating agents can induce the expression of these antigens, allowing a reaction by engineered lymphocytes suggesting that epigenetic therapy can be combined with immunotherapy for better results (41).

Currently available DNA methylation inhibitors lead to overall DNA methylation inhibition. Global hypomethylation may lead to the activation of oncogenes and/or increased genomic instability. Recently, DNA methylation inhibitors that targeted specific genes or the groups of genes are being developed. Another way is to identify and use demethylases.

DNA methylation is involved in key cellular processes important to tissue-specific gene expression, cell differentiation, and development. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer (44). It is largely unknown about the mechanisms that control methylation levels and prevent from DNA methylation at CpG islands. After fertilization and at certain points of the embryonic development, a large number of the methylation markers are erased (45). Importantly, despite an intense search, no direct DNA demethylase has been identified yet (44). Until recently, the only known epigenetic marker of DNA itself was 5mC, which is converted by DNA methyltransferases preferentially at CpG dinucleotides (46). 5-Hydroxymethylcytosine (5hmC), which is considered to be the sixth base of the genome of higher organisms, has recently been identified (45). The recent discovery that the 3 members of the TET protein family can convert 5mC into 5hmC has provided a potential mechanism leading to DNA demethylation (44).

(25)

25

Figure 3.4.1. Demethylation model

The canonical DNA nucleosides dG, dA, dT, and dC. Cytosine can be modified to mC and hmC in mammalian tissues. hmC could be further oxidized to the putative demethylation intermediates fC and caC, but have never been found in vivo (45).

Despite a lot of promising epigenetic data, they have not been used in routine diagnostic practice yet. The hypermethylation of gene promoters may be considered the tip of iceberg of deregulation of complex epigenetic mechanisms that contribute to GBM onset (19). Whole genome, transcriptome, and methylome sequencing is currently at the horizon of clinical research and could be performed even in small-sized tumor tissue samples (47). Unraveling the epigenetic alterations opens up possibilities for discovering new biomarkers for cancer detection and prognosis (19). Restoring epigenetically altered pathways will probably lead to the development of new therapeutic tools in GBM.

Blood plasma in cancer patients contains DNA derived from cells due to necrotic or apoptotic cancer cells releasing genomic DNA. This potentially provides a less invasive method for the detection of biomarkers. Aberrantly methylated cancer genes found in plasma could be one such type of biomarkers (33).

3.5.Genes selected for more detailed study

The cytosine methylation of CpG dinucleotides in gene promoters is a common cause of DNA silencing and transcriptional repression that can

(26)

26

modulate the clinical characteristics of glioblastoma. The best known is MGMT promoter methylation determining tumor response to DNA alkylating agents and being an independent prognostic factor for patients’ survival (48). However, several widely described genes can be seen as only a partial picture of the methylation changes, and there may be many more genes that need to be clarified. Recent methylome studies of brain tumors have disclosed a list of new epigenetically modified genes associated with gliomagenesis and different glioma clusters (2, 6, 8). The latter studies illustrate a glioblastoma profile being constructed via methylation of a multiple set of genes, forming networks attributed to different pathogenesis pathways. It has been suggested that genes, such as AREG, CASP8, CD81, DcR1, DR4, GATA4, GATA6, hMLH1, NPTX2, TES, and TFPI2 could be important in gliomagenesis.

Table 3.5.1. Functions and reported methylation frequency of the selected genes in glioblastoma

Gene Location Functions of encoded proteins GBM methylation in %, (literature source) AREG 4q13

Autocrine growth factor and mitogen for astrocytes, Schwann cells, and fibroblasts

ND

CASP8 2q33

Responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced apoptosis

10-30 (49, 50)

CD81 11p15.5

Encoded protein is a cell surface glycoprotein that is known to form a complex with integrins

54 (2, 6)

DcR1 8p22

May protect cells against TRAIL mediated apoptosis by competing with TRAIL-R1 and R2 for binding to the ligand

ND

DR4 8p21 Receptor for the cytotoxic ligand

TNFSF10/TRAIL 25-70 (2, 26, 49) GATA4 8p23.1 Transcriptional activator ND

GATA6 18q11.1 Transcriptional activator 30-48 (2, 50-52) hMLH1 3p21.3 Modulates postreplicative DNA

mismatch repair system 0-15 (53, 54) NPTX2 7q21.3 Modulates long-term cell plasticity ND

TES 7q31.2

May play a role in cell adhesion, cell spreading and in the reorganization of the actin cytoskeleton

58-69 (2, 7)

TFPI2 7q22

May play a role in the regulation of plasmin-mediated matrix

remodeling

ND ND, no data on DNA methylation in glioblastomas.

(27)

27

AREG

The AREG (amphiregulin, SDGF, schwannoma-derived growth factor) gene is located on chromosome 4q13.3. AREG is a member of the epidermal growth factor family. It is a bifunctional growth modulator: it interacts with the EGF/TGF receptor to promote the growth of keratinocytes and normal lung epithelial cells in the presence of extracellular matrix, but inhibits the growth of certain aggressive carcinoma cell lines, some transformed cell lines, and lung epithelial cells in the absence of extracellular matrix (55, 56). Thus, AREG can either stimulate or inhibit the growth of lung cancer cells, depending on the biological settings (56). Various studies have highlighted the functional role of AREG in several aspects of tumorigenesis, including self-sufficiency in generating growth signals, limitless replicative potential, tissue invasion and metastasis, angiogenesis, and resistance to apoptosis (57). On the other hand, there is an growing body of data about AREG promoter methylation in various types of tumors: gastric (58), high-grade serous carcinomas (59), and human bladder tumor cell line (T24) (60). AREG is differentially methylated in low-grade gliomas (WHO grade II) in comparison with controls (6). Microarray analysis coupled with the pharmacologic demethylation of cultured tumor cells is a useful means for investigating genome-wide epigenetic events and has been successfully used for this purpose in glioblastoma; one of the novel candidate genes identified by this approach is AREG (7).

CASP8

The CASP8 (caspase 8, apoptosis-related cysteine peptidase 8) gene is located on chromosome 2q33.1. This gene encodes a member of the cysteine-aspartic acid protease (caspase) family. The sequential activation of caspase 8 plays a central role in the execution phase of cell apoptosis (Figure 3.5.1). Caspases exist as inactive proenzymes composed of a prodomain, a large protease subunit, and a small protease subunit. The activation of caspases requires proteolytic processing at conserved internal aspartic residues to generate a heterodimeric enzyme consisting of the large and small subunits. This protein is involved in the programmed cell death induced by Fas and various apoptotic stimuli. The N-terminal FADD-like death effector domain of this protein suggests that it may interact with Fas-interacting protein FADD (61).

A significant epigenetic silencing of the proapoptotic CASP8 gene was observed during the progression of primary to recurrent GBM, probably conferring tumor cells a relevant further growth advantage. In brain tumors, CASP8 is hypermethylated in 40% of neuroblastomas and medulloblastomas. Moreover, a high concordance between gene

(28)

28

methylation, decreased mRNA levels, and protein expression was observed as well (26).

Figure 3.5.1. Apoptotic TRAIL signaling

Binding of TRAIL to death receptors (DR4, DR5) leads to the recruitment of the adaptor molecule, FADD. DcR1/2 work as inhibitors of DR4/5. Pro-caspase-8 binds to FADD leading to DISC formation and resulting in its activation. Activated caspase-8 directly activates executioner caspases (caspase-3, -6, and -7) (type I cells) or cleaves Bid (type II cells). Translocation of the truncated Bid (tBid) to the mitochondria promotes the assembly of Bax-Bak oligomers and mitochondria outer membrane permeability changes. Cytochrome c is released into cytosol resulting in apoptosome assembly. Active caspase-9 then propagates a proteolytic cascade of effector caspases activation that leads to morphological hallmarks of apoptosis. Further cleavage of pro-caspase-8 by effector caspases generates a mitochondrial amplification loop that further enhances apoptosis. When FLIP levels are elevated in cells, caspase-8 preferentially recruits FLIP to form a caspase-8-FLIP heterodimer which does not trigger apoptosis (64).

A study by Qi et al. showed the heterogeneity of glioblastomas and derived cancer stem cells in the genomic status of CASP8, expression of CASP8, and thus responsiveness to TRAIL-induced apoptosis. Clinic trials may consider genomic analysis of the cancer tissue to identify the genomic

(29)

29

loss of CASP8 and use it as a genomic marker to predict the resistance of glioblastomas to TRAIL apoptosis pathway-targeted therapies (62).

The loss of CASP8 expression in cancer cells may result in resistance to drug-induced apoptosis and may be important for a therapeutic strategy. Recent studies have demonstrated that the demethylation of CpG islands of the CASP8 gene in resistant cancer cells sensitizes them to chemotherapeutic agents (63). Moreover, the inactivation of CASP8 expression by promoter hypermethylation may result in cancer progression.

COX7A1

The COX7A1 (cytochrome c oxidase subunit VIIa polypeptide 1) gene is located on chromosome 19q13.1. The product of this gene is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport (70). COX catalyses the electron transfer from reduced cytochrome c to oxygen, producing H2O. This

reaction is coupled with proton transfer from the matrix to the intermembrane space, thereby contributing to the energy stored in the electrochemical gradient. The catalytic core of COX is composed of mitochondrial encoded proteins whereas nucleus-encoded proteins contribute to the structural and regulatory subunits of the complex. COX7A1 promoter methylation has been correlated with gene expression (71). COX7A1 demonstrated increased expression after 5-azadc treatment in methylated breast tumor cell lines (72). In an independent cohort of breast tumor/normal pairs, COX7A1 demonstrated increased methylation in 70.6% (12/17) of tumors compared with a corresponding normal tissue, and a further 5 showed the equal levels of low-level methylation in a tumor and a corresponding normal tissue (72). This gene has been detected differentially expressed in lung cancer (73).

CD81

The CD81 (CD81 molecule, TAPA-1) gene is located on chromosome 11p15.5. This gene is localized in the tumor-suppressor gene region, and thus it is a candidate gene for malignancies (65). It encodes a cellular membrane protein belonging to the tetraspanin family. Most of these members are cell-surface proteins that are characterized by the presence of 4 hydrophobic domains. This encoded protein is a cell surface glycoprotein that is known to form a complex with integrins. The protein complex mediates the events of signal transduction that play a role in the regulation of cell development, activation, growth, and motility. In addition, CD81

(30)

30

may be involved in signal transduction. There are data that CD81 tetraspanin may have an inhibitory effect on cell movement and metastases (Figure 3.5.2) (66, 67). The gene was found to be silenced by methylation in multiple myeloma cell lines (68). Recent glioblastoma methylome studies have shown a CD81 methylation rate of 54% (2, 6).

Figure 3.5.2. Tetraspanin signaling

CD81 (and CD9) is associated with phosphatidylinositol 4-kinase (PI4K), which locally produces phosphoinositides (such as phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)). This causes the recruitment and activation of SHC

(SRC-homology-2-domain-containing transforming protein). Subsequent Ras-mediated activation of extracellular signal-regulated kinase (ERK) or p38 or Jun N-terminal kinase (JNK) pathways leads to proliferation or apoptosis, respectively. Caspase-3 activation might also contribute to CD9-dependent regulation of apoptosis. Signaling through CD151 might negatively regulate Ras-ERK/MAPK and Akt/protein kinase B (PKB) signaling in a cell-adhesion-dependent manner. CD151 (together with associated laminin-binding integrins) also preferentially activates Rac (and Cdc42) over Rho, leading to regulation of the actin cytoskeleton, cell spreading and motility. Conversely, CD82 might suppress tumor cell invasion by downregulating p130Cas and thereby decreasing Rac activation. The C-terminal tails of CD151 (and CD81and CD9) contain PDZ-domain-binding motifs, and the tail of CD151 is essential for its signaling and adhesion-strengthening functions. Possibly, PDZ-domain-containing proteins could connect these tetraspanins to signaling and cytoskeletal regulatory events that affect adhesion strengthening, cell spreading and cell motility (69) .

(31)

31

DcR1

Despite 8p being a relatively small chromosome arm, it is one of the most frequently altered genomic regions in human cancer, and is also rich in candidate oncogenes and tumor suppressor genes associated with the development of certain types of cancers (74). Deletions in this region are observed in glioblastoma (75). One of these genes located on chromosome 8p region is DcR1 (tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain, TNFRSF10C). The protein encoded by the DcR1 gene is a member of the TNF-receptor superfamily. DcR1 lacks the intracellular death domain, can bind TRAIL, but is not capable of transmitting the death signal because of its defective death domains (76). This receptor is not capable of inducing apoptosis and is thought to function as an antagonistic receptor that protects cells from TRAIL-induced apoptosis. The expression of this gene was detected in many normal tissues but not in most cancer cell lines, which may explain the specific sensitivity of cancer cells to the apoptosis-inducing activity of TRAIL (77). DcR1 promoter methylation was observed in 40.4% of primary breast tumors (78). Also, high methylation levels at the DcR1 promoter region in peripheral blood was positively associated with perineural tumor spread and shorter patients’ survival (79). DcR1 was frequently hypermethylated in hepatocellular carcinoma tissue samples, and its methylation was found to be closely associated with the inactivation of gene expression (80). As an alternative to a deletion, DcR1 promoter methylation may be associated with clinical data of glioblastoma patients. The methylation of the DcR1 gene promoter was determined in 21% of low-grade gliomas (6) suggesting its role in gliomagenesis.

TRAIL acts through 2 pairs of opposing receptors, 2 proapoptotic death receptors, DR4 and DR5, and 2o antiapoptotic decoy receptors, DcR1 and DcR2. All TRAIL receptors have been mapped to the same chromosomal locus, 8p21-22, suggesting evolution from a common ancestral gene (76).

DR4

The gene DR4 (TNFRSF10A, tumor necrosis factor receptor superfamily, member 10a) is located on chromosome 8p21. DR4 is a receptor for the cytotoxic ligand TNFSF10/TRAIL. The adapter molecule FADD recruits Casp8 to the activated receptor. The resulting death-inducing signaling complex (DISC) performs Casp8 proteolytic activation, which initiates the subsequent cascade of caspases (aspartate-specific cysteine proteases) mediating apoptosis (81). The methylation of the DR4 gene promoter was found to be extremely common, in both early gastric carcinomas and

(32)

32

advanced gastric carcinomas (98.4% and 83.3%, respectively) (76). rhTRAIL and 2 newly designed monoclonal antibodies targeting DR4 (mapatumumab and HGS-ETR1) are new promising cancer drugs because these agents have been shown to preferentially induce apoptosis in tumor cells. However, the main obstacle in TRAIL-based therapies remains the resistance of cancer cells to TRAIL and its analogues. Therefore, identifying the resistance mechanisms and the predictors of response in these tumors remains a key issue in modern cancer diagnostics and therapeutic strategies. Among other possibilities, such predictive factors may be epigenetically regulated in a tumor-specific manner (49). DR4 promoter methylation is frequent in human astrocytic gliomas, and the epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies (49). The hypermethylation of DR4 (up to 66.7%) was observed in neuroblastomas (82). In non-CNS malignancies, higher methylation rates of DcR1 and DcR2 were reported (up to 100%), whereas DR4 and DR5 were rarely hypermethylated (83). In contrast to the findings reported by van Noesel et al. and Shivapurkar et al., Martinez et al. did not observe the CpG island hypermethylation of the potentially antiapoptotic DcR1 in any GBM as well as the lower rates of hypermethylation of DR4 and DR5 (25 and 12.5%, respectively) (26). The development of targeted therapies restoring functional extrinsic apoptosis, as recently shown in vivo with the synergistic combination of the DNA demethylating agent decitabine and TRAIL, may provide a useful tool to overcome the resistance of GBM to contemporary treatment modalities.

GATA4

The GATA4 (GATA binding protein 4) gene is located on chromosome 8p23.1. This gene encodes a member of the GATA family of zinc-finger transcription factors. The members of this family recognize the GATA motif, which is present in the promoters of many genes (84). GATA4 regulates the gene expression of various genes, which are involved in cardiac development, cardiac hypertrophy, and heart failure. In addition to the heart, GATA4 plays important roles in the reproductive system, gastrointestinal system, respiratory system, and cancer. Therefore, the positive and negative regulations of GATA4 are important components of biologic functions (85). Recently, GATA4 expression in normal embryonic and adult mouse and human astrocytes has been reported, in which it functions as an inhibitor of proliferation and inducer of apoptosis (86). GATA4 knock-out in mice lead to embryonic lethality due to cardiac defects (87, 88), and GATA4 mutations cause Holt-Oram syndrome and congenital

(33)

33

heart defects. GATA4 is frequently silenced in lung, colon, prostate, ovarian, and breast cancer (89-93), but its exact role in cancer biology and the mechanisms by which it operates are poorly understood (94). The expression of GATA4 was extinguished in the majority of cell lines from colorectal and gastric cancers as well as in primary tumors. Silencing was associated with the hypermethylation of the GATA4 promoter sequences. Moreover, the candidate target genes of the GATA4 factor were commensurately decreased in their expression and also displayed the methylated promoters (95).

GATA4 loss occurred through promoter hypermethylation or novel somatic mutations (94). The re-expression of GATA4 also conferred the sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in the GBM therapy (94). GATA4 can be activated through stimulating the activity of the already existing GATA4 protein molecule or by increasing the GATA4 protein expression. GATA4 protein expression can be altered through transcriptional or translational mechanisms (85).

Given the role of GATA4 in regulating astrocyte proliferation and the observed loss of GATA4 in several human cancers, GATA4 was demonstrated to be a novel tumor suppressor in GBM.

GATA6

The GATA6 (GATA binding protein 6) gene is located on chromosome 18q11.2. GATA6 is one of 6 members of the mammalian GATA family of transcription factors, all containing 2 highly conserved zinc-finger DNA binding domains that interact with a canonical DNA motif (G/A)GATA(A/T) (96). It is thought that GATA6 may be important for regulating terminal differentiation and/or proliferation. It is also linked to embryonic cardiac development. The loss of GATA6 resulted in the enhanced proliferation and transformation of astrocytes (52). Knockin GATA6 expression in human malignant astrocytoma cells reduced their tumorigenic growth with decreased VEGF expression. The disruption of GATA6 led to transformation and demonstrated that GATA6 is a new and relevant human GBM tumor suppressor gene (52). The great majority of human glioblastomas (which can arise from low-grade astrocytomas), but not low-grade astrocytomas, displayed the loss of GATA6 expression, mutations in GATA6, and loss of heterozygosity. The re-expression of GATA6 in human malignant astrocytoma cells inhibited their growth (52). This establishes GATA6 as a bona fide tumor suppressor in this disease, marking the progression from low-grade astrocytoma to malignant glioblastoma (95).

(34)

34

hMLH1

The hMLH1 (MutL homolog 1, colon cancer, nonpolyposis type 2 E. coli) gene is a human gene located on chromosome 3p21.3. hMLH1 plays a role in DNA mismatch repair. hMLH1 heterodimerizes with PMS2 to form MutL alpha, a component of the postreplicative DNA mismatch repair system (MMR) (Figure 3.5.3) (97).

Figure 3.5.3 Mismatch repair

A mispaired base is recognized by the hMSH2/GTBP complex while an insertion/deletion loop is recognized by the hMSH2/hMSH3 complex. MutL related proteins (hMLH1/hPMS2 and hMLH1/hPMS1 complexes) then interact with the MutS related proteins that are already bound to the mispaired bases. The hMSH2/GTBP complex may also support the repair of insertion/deletion loops (97).

DNA repair is initiated by MutS alpha (MSH2-MSH6) or MutS beta (MSH2-MSH6) binding to a dsDNA mismatch, and then MutL alpha is recruited to the heteroduplex. The assembly of the

(35)

MutL-MutS-35

heteroduplex ternary complex in the presence of RFC and PCNA is sufficient to activate the endonuclease activity of PMS2. It introduces single-strand breaks near the mismatch and thus generates new entry points for the exonuclease EXO1 to degrade the strand containing the mismatch. DNA methylation would prevent cleavage and therefore assure that only the newly mutated DNA strand is going to be corrected. MutL alpha (MLH1-PMS2) interacts physically with the clamp loader subunits of DNA polymerase III, suggesting that it may play a role to recruit DNA polymerase III to the site of the MMR. Also implicated in DNA damage signaling, a process, which induces cell cycle arrest and can lead to apoptosis in case of major DNA damages. hMLH1 heterodimerizes with MLH3 to form MutL gamma, which plays a role in meiosis (98). hMLH1 inactivation by promoter hypermethylation has been reported to be associated with some human cancers (99-101). Herman et al. have suggested that DNA methylation associated with the transcriptional silencing of hMLH1 is the underlying cause of mismatch repair defects in most sporadic colorectal cancers (102). Xinarianos et al. have shown that 58.6% and 57.8% of lung cancer tumor specimens had the reduced expression levels of the hMLH1 and hMSH2 proteins, respectively (103).

KRT81

The KRT81 (keratin 81, also KRTHB1 or Hb-1) gene is located on chromosome 12q13. The protein encoded by this gene is a member of the keratin gene family. As type II hair keratin, it is a basic protein that heterodimerizes with type I keratins to form hair and nails (104). Keratins are proteins expressed in all types of epithelial cells (105), with different expression patterns among different carcinomas (106), and they are extensively used as diagnostic markers. To date, KRT81 has not been validated as a prognostic marker, and the present study is the first to link a KRT81 variant to tumor recurrence. Campayo et al. have found that KRT81 may be a new immunohistochemical marker of squamous cell carcinoma (107). KRT81 showed a clear positive staining in squamous cell carcinomas (95% positive), whereas 81% of adenocarcinomas were negative (107). Their findings indicate that single nucleotide polymorphisms in KRT81 and XPO5 could be proved to be useful biomarkers for individualizing the therapy in patients with non–small cell lung cancer and that KRT81 may be a novel immunohistochemical marker of squamous cell carcinoma, providing a new diagnostic tool to be used in therapeutic decision-making (107).

A STUDY OF TUMOR SUPPRESSOR GENE EXPRESSION AND PROMOTER METHYLATION FOR THE IDENTIFICATION OF PROGNOSTIC MARKERS IN GLIOBLASTOMA

Paulina Vaitkienė