Cancer Epigenetics (Biomolecular Therapeutics for Human Cancer) || Epigenetics in Pediatric Cancers

9EPIGENETICS IN PEDIATRICCANCERS

Roberta Ciarapica, Lavinia Raimondi, Federica Verginelli,and Rossella RotaLaboratory of Endothelial Cells and Angiogenesis, Ospedale Pediatrico Bambino Gesu,Rome, Italy

9.1 INTRODUCTION

Epigenetics is the newest research front to unravel the nuanced world of geneexpression. Gene expression can be epigenetically regulated through the cooper-ation of multiple processes such as DNA methylation, histone proteins modifi-cation, histone variants replacement, rearrangement of nucleosomal positioning,and mechanisms involving noncoding small RNAs. As largely explained in theprevious chapters, the term “epigenome” encompasses those heritable states ofgene expression that are not associated with sequence changes in DNA. Oncethe epigenetic imprinting is established during embryogenesis and development,it is maintained throughout cell division. Unlike the genome, the epigenomeis far from being identical in all the diverse cell types of an organism. Theepigenome is instead dynamic and reprogrammable in the context of even onesingle cell. In order to define the epigenome, two principal components haveto be considered: the chromatin structure and the pattern of DNA methylation.Owing to the extensive research in the last decades, chromatin is no more con-sidered a static and merely structural compartment of the cell, but it is viewedas a very complex entity that is able to regulate gene expression. It is throughthe histone code, the overall covalent modifications of histones, that biological

Cancer Epigenetics: Biomolecular Therapeutics for Human Cancer, First Edition.Edited by Antonio Giordano and Marcella Macaluso.© 2011 Wiley-Blackwell. Published 2011 by John Wiley & Sons, Inc.

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164 EPIGENETICS IN PEDIATRIC CANCERS

processes from the chromatin remodeling up to DNA repair, replication, and seg-regation are regulated. Histones can be acetylated, methylated, phosphorylated,ubiquitinated, and sumoylated (Strahl and Allis, 2000). Moreover, the combi-nation of these modifications gives rise to molecular platforms recognizable bytransacting factors that interact with acetylated histones and methylated lysines.An additional regulation level is dictated by the presence of histone variantsthat, changing the nucleosome stability and mobility, cooperate in modifyinggene-expression pattern. Besides chromatin remodeling, which alters the acces-sibility of the genes to the transcription machinery, epigenetic networks imparttheir imprinting through DNA methylation. This last event results from bothde novo methylation of unmethylated DNA and maintenance of methylation ofhemimethylated sequences. The addition of the methyl group is catalyzed byDNA methyltransferases (DNMTs) generally at the level of cytosines withinCpG-rich regions, named CpG islands . DNA hypermethylation of promoter CpGislands is a mechanism to silence transcriptionally active genes. This kind ofmethylation represents a regulatory mechanism during normal cell development.The reaction is catalyzed by DNA methyltransferases capable of converting cyto-sine into 5-methylcytosine. Because of its susceptibility to deamination, thismodified residue becomes a frequent mutation point, leading to carcinogenesisor to epigenetic reprogramming depending on the cellular context (Morgan et al.,2004). In addition, areas of hypermethylation have been correlated to chromoso-mal aberrations. Even if most of the methylated CpG islands are located withinrepetitive elements of heterochromatin, several other CpG islands have been iden-tified in euchromatin within the regulatory elements of genes. Gene silencing byDNA methylation can be established either at the level of recognition sequencesimpairing the binding of transcription factors, or recruiting methyl-CpG bindingdomain proteins (MBD) that exert an adaptor role between methylated DNA andchromatin-modifying enzymes. Recently, it has been observed that DNA methyla-tion can be affected by noncoding RNAs such as small interfering RNA (siRNA).In particular, RNA molecules have been implicated in guiding DNA methyla-tion at the level of CpG islands. Interestingly, siRNA-directed DNA methylationhas been reported in human cells to lead to transcriptional gene silencing (TGS)(Astuti et al., 2004b). All the data collected till now about the epigenetic controlof gene expression support the concept of a strong interconnection among all theepigenetic mechanisms regulating the access of specific factors to gene promoters.

Alterations of these fine mechanisms lead to aberrant expression of genesinvolved in proliferation, cell cycle, apoptosis, and differentiation, and, in asso-ciation with genetic mutations, can contribute to the development and progressionof cancer. Gene silencing is a fundamental biological process necessary for theproper development of eukaryotic organisms. Differentiation, imprinting, andX-chromosome condensation are some of the most important phenomena thatneed chromatin remodeling (i.e., condensation) through covalent modificationsof histones and cytosine methylation. The established pattern of gene silencingis maintained in specific subsets of cells because of the heritability of epigeneticmodifications through mitosis. The deregulation of epigenetic silencing results

INTRODUCTION 165

in multiple changes that are called epimutations that, like genetic mutations,may confer a selective advantage to replicating cells. As the most frequent areasfound hypermethylated in cancer are DNA regions containing tumor-suppressorgenes (TSGs), hypermethylation is considered to be a hallmark of human tumors,although promoter hypermethylation by itself does not demonstrate suppressionof gene function (Esteller et al., 2001).

Notwithstanding the importance of aberrant areas of DNA hypermethylationin cancer cells, the global loss of DNA methylation is the most typical char-acteristic of a cancer epigenome. Hypomethylation of CpG islands at the levelof promoter regions can increase gene expression, sometimes activating proto-oncogenes. Nevertheless, the overall decrease in the level of 5-methyl cytosineprevalently affects the intergenic and intronic sequences of DNA, resulting inchromosomal instability and mutations. This general demethylation is assumedto activate the normally dormant transposons and endogenous retroviruses thataccount for almost half of the human genome, thus fostering further mutations andchromosomal recombinations. Considering that some CpG islands become methy-lated with age (Xue Jun et al., 2003) and probably with tissue differentiation,cancer-associated hypomethylation confers a more immature gene expression pro-file to the affected cells, resembling that of a stem cell, but with problems ofgenome instability. One of the striking examples in this regard is the alteration ofthe methylation pattern of imprinted genes by loss of imprinting (LOI). Genomicimprinting is a typical phenomenon in mammals. It represents a form of non-Mendelian inheritance in animals, where the “imprinted” genes are uniquelyexpressed from one allele. The expressed allele, either paternal or maternal, ismaintained throughout the successive generations of cells of a tissue, unless agenetic or epigenetic alteration has occurred (Jelinic and Shaw, 2007). Approx-imately 40 imprinted genes have now been identified in the human genome,with roles in prenatal growth and development of particular cell lineages, and inhuman diseases.

LOI can derive from either hypomethylation of alleles that are normally notexpressed or hypermethylation of alleles that are normally expressed or of theirregulatory sequences. Aberrant methylation of imprinted genes results in LOI andconsequently in biallelic expression of genes that can support cancer developmentand concomitant repression of TSGs. One of the well-known events of LOIoccurs at the IGF2-H19 locus in pediatric cancer and in children’s overgrowthdiseases predisposing to cancer, including the Beckwith–Wiedemann syndrome(BWS).

Leukemia, central nervous system (CNS) tumors, and sarcomas are the mostfrequent cancers in children. The percentage of primary tumors by tumor siteof origin can vary with age (Fig. 9.1). Pediatric cancers markedly differ fromadult malignancies in their nature, distribution, and prognosis. Children’s tissuesare less exposed to environmental tumorigenic factors as opposed to adulttissues, suggesting an implication of host intrinsic factors in pediatric cancers.In support of these theories, it has been observed that the methylation patternsof a cell change in an age-related manner. However, even if both hypo- and


5–8 Years

Brain(25%)

Other(10%)

Softtissue(5%) Eye

(2%)

Kidney(5%)

Bone(3%)

Neuroblastoma(3%)

Lymphoma(16%) Acute

leukemia(31%)

<5 Years

Lymphoma(10%)

Brain(13%)

Other(9%)

Softtissue(7%) Eye

(6%) Kidney(10%) Neuroblastoma

(7%)

Acuteleukemia

(36%)

Ovary/testis(2%)

10–14 Years

Lymphoma(25%)

Brain(18%)

Other(16%)

Softtissue(5%)

Bone(11%)

AcuteLeukemia

(18%)

Ovary/testis(3%)

Thyroid(4%)

15–19 Years

Softtissue(5%)

Eye(4%)

Lymphoma(27%)

Brain(10%)

Other(10%)

Bone(7%)

Acuteleukemia

(12%)

Ovary/testis(11%)

Thyroid(8%)

Melanoma(6%)

FIGURE 9.1 Principal pediatric cancers: from birth to 19 years old. (Behrman et al.,2000.)

hypermethylation accumulate with age, leading to increased risk of cancerwith advancing age in elderly people, it is clear that this is not the case inpediatric neoplasia. In other words, whereas in adult tissues these events canalso be part of the aging process, in childhood cancers methylation changesnotably represent developmental deregulation. In agreement with this theory,one of the most striking features of most of the pediatric cancers is that theyarise from embryonic-derived cells and not from terminally differentiated tissuecells. These immature cells are likely to develop dysfunctions during theirnormal differentiation. To unravel the causative factors in the early stage ofcancer development, the old concept that even a single tumor is characterizedby heterogeneous population of cells, among which some are responsible forthe highest tumorigenic potential, has been replaced by the hypothesis of cancerstem cells. Even if this idea is controversial, a lot of researchers believe thatdifferentiation abnormalities occur in cells capable of self-renewal, leading to thedevelopment of cancer. This is an intriguing theory especially in an attempt tounravel the molecular mechanisms that give rise to embryonal pediatric cancer.

One of the features of these tumors is the cell heterogeneity within thetumor. It has been hypothesized that epigenetic changes account for the different

INTRODUCTION 167

phenotypes of the cell populations constituting a single tumor mass, as itoccurs in neuroblastoma (NB), one of the most aggressive solid pediatriccancers. It is possible that the altered epigenetic pattern believed to cooperatein the pathogenesis of NB could be spontaneously reverted in those rare andage-dependent forms that undergo spontaneous regression, reflecting activationof an apoptotic/differentiation program.

Unlike adults, the major problem in the treatment of pediatric cancers isthe adverse effect of treatment on the growth and development of the patient.Targeting aberrant patterns of methylation represents a promising anticancerstrategy in view of the reversibility of this DNA modification. Several clini-cal trials involving modulators of methylation are ongoing (Szyf et al., 2004;Garcia-Manero et al., 2009). An important consequence of epigenetic studies isthe identification and development of novel compounds used, in association withconventional chemotherapy, to induce growth arrest, differentiation, and tumorcell death. It has been demonstrated that histone deacetylase inhibitors (HDACi)(such as VPA, valproic acid and TSA, thricostatin A) and inhibitors of the DNMTenzymes (such as 5-AzaC and decitabine) have antitumor potential, reactivatinggene expression in cancerous cells (Bhalla, 2005). Moreover, new studies aim toproduce antileukemic siRNAs in order to target and block fusion transcripts onlyin leukemic cells without damaging normal cells (Thomas and Kansara, 2006).Clinical studies indicate that the use of these novel compounds seems to exerttherapeutic benefits in patients with leukemia (Galm et al., 2006), even if thesetreatments could lead to the reexpression of genes not directly correlated withcancer. To avoid this aspecific gene reactivation, it could be useful to gener-ate more targeted therapies. From this viewpoint, the future epigenetic therapycould include an additional tailored medicine on the basis of the overwhelmingcollection of data obtained from epigenotype profiling studies.

It is now clear that an increasing number of human pathologies can be cor-related with aberrant epigenetic mechanism, including cancer, neurodegenerativesyndromes, and imprinting disorders. A deeper knowledge of these perturbed, butalso reversible, epigenetic events may provide the molecular basis for the devel-opment of new and effective pharmacological compounds that aim to disruptspecifically diseased cells.

This section focuses on recent discoveries concerning perturbations of epi-genetic mechanisms in pediatric cancers. It is noteworthy that few studies areavailable in the field on young patients, especially on uncommon pediatric can-cers, because of the relatively small number of cases in the population comparedto adult cases. To further complicate the subject, results from some studies can besometimes conflicting because of the following factors: (i) different methods ofdetection whose results are often dependent on assay conditions; (ii) small cohortof primary tumors (<20); (iii) inaccessibility of normal counterparts for compara-tive studies (i.e., nonneoplastic cells as for ependymal cells); and (iv) complexityin accurately defining the degree of gene methylation. In addition, in most of thestudies gene silencing was not confirmed in tissues by gene-expression detectionthrough RT-PCR (reverse transcriptase-polymerase chain reaction) because of the


inability to obtain adequate RNA samples. Finally, heterogeneity of cells withina tumor mass can contribute to controversial findings, as reported for infiltratingleukocytes as a source of methylation (Lombaerts et al., 2004).

Nevertheless, epigenetic studies have led the way to understanding the finemechanisms of gene regulation.

9.2 LEUKEMIA IN CHILDHOOD

Leukemia represents the most common cancer in children and accounts forabout one-third (30%) of all pediatric cancers. In particular, acute lymphoblasticleukemia (ALL) represents approximately 75% of all leukemia cases, and thepeak incidence is at the age of four. Acute myeloid leukemia (AML) accountsfor about 20% of leukemia cases and has an incidence that is considered to bestable between birth and the age of 10. Finally, chronic myeloid (CML) andchronic lymphoid (CLL) forms are rarely observed in children (Klose et al.,2007). Every type of leukemia can be subdivided into various subtypes, accord-ing to morphologic, immunologic, cytogenetic, and molecular genetic features ofleukemia cells, and every phenotype has both prognostic and therapeutic impli-cations. Although all of these leukemia subtypes show similar clinical features,also because they involve disruption of bone marrow function, their response totherapy can vary in a significant way. Chromosomal translocations are present inat least 80% of childhood leukemia. In particular, these chromosomal transloca-tions involve genes required for hematopoiesis, thus interfering with the correctcell differentiation process. As a matter of fact, a fundamental characteristic ofthis malignant disease is that mechanisms involving proliferation, differentiation,and apoptosis are deregulated in hematopoietic cells. Most of the genetic aberra-tions are well defined in leukemia, and it has been demonstrated that the genesisof fusion proteins, as a result of chromosomal breaks and rearrangements, isdirectly connected with the arrest of differentiation at a specific stage. Besidesthese very peculiar genetic aberrations, recent data provide evidence to attest theimportance of epigenetic aberrations, such as DNA hyper/hypomethylation andhistones ace/deacetylation, in modifying the expression pattern of a great numberof genes either generally involved in the pathogenesis of leukemia or in the gen-esis of fusion proteins itself. In many childhood acute leukemias, transcriptionfactors are aberrantly altered in their function by chromosomal rearrangements,thus modifying important epigenetic mechanisms and leading to malignant trans-formation (Berman and Look, 2007).

As shown in Fig. 9.2, chimeric proteins, such as AML1- ETO and PML-RARα, can alter the normal function of the hematopoietic transcription factor(HTF) in the regulation of target genes. It seems that fusion proteins show astronger affinity for corepressor proteins, histone deacethylases (HDACs), andDNA methyl transferases (DNMTs), thereby repressing gene expression.

Epigenetic modifications may contribute in the earliest stages of neoplasia byaffecting the transcription of genes involved in hematopoietic precursor differen-tiation and lineage specification (Esteller, 2007; Zardo et al., 2008).

LEUKEMIA IN CHILDHOOD 169

ActivationOFF

OFF

ON

Abberant silencingin leukemic cells

Regulation

Chromosomal transioncation(e.g., PML.RAR. PLZF-RAR AML 1-ETO)

(e.g., RA)

Repression

FIGURE 9.2 Normal and pathological function of the hematopoietic transcription factor(HTF) in the regulation of target genes: role of the fusion proteins. (From Di Croce, 2005.)(See insert for color representation of the figure.)

Large fragments of DNA are abnormally methylated in leukemia cells anddifferent degrees of methylation are correlated with distinct levels of gene silenc-ing. In particular, similar to other tumor cells, leukemia cells exhibit globalhypomethylation of the genome accompanied by region-specific hypermethyla-tion events. In fact, the DNA from patients affected by chronic lymphocyticleukemia (CLL) is globally hypomethylated when compared with normal cells(Esteller et al., 2001). As global hypomethylation in both intronic and codingregions may contribute to genomic instability with consequent increased risk ofDNA deletions, it is believed to foster the genesis of fusion proteins in leukemias.

Finally, in some leukemias an aberrant activity of the two classes of enzymesthat mediate the acetylation and deacetylation of specific histones, histone acetyltransferases (HATs) and HDACs, has been observed.

Here, we review the role of epigenetic changes in leukemogenesis; in fact,recent findings have demonstrated that aberrant DNA methylation, histoneacetylation, and synthesis of fusion proteins can really contribute to diseasedevelopment.

9.2.1 Genesis of Fusion Proteins and Aberrant Acetylation in Leukemia

Several studies indicate that chromosomal translocations of genes whoseexpression is critical to hematopoiesis processes are critical passages towardthe leukemic phenotype. These DNA breaks and insertions generate leukemicfusion oncoproteins, which are constituted by portions of two differenttranscription factors. An interesting model suggests that these fusion proteinshave leukemogenic potential but, alone, are not enough to cause cancer;cooperation with other genetic mutations leads to clonal expansion, blocking ofhematopoietic precursor differentiation, and leukemogenesis (Zardo et al., 2008).


Fusion proteins have a strong leukemogenic potential and are, in turn, involvedin other epigenetic events. The production of chimeric proteins is typical of agreat number of leukemias, also because the “permissive” cellular context ischaracterized by a high frequency of recombination events (Vire et al., 2006).

Many translocations have been described in both childhood and adultleukemias; some of these are typical, or more frequent, in one of them, whileothers have been found in both of them.

Gene expression profiling can identify genetic subtypes that are prognosticallyimportant for leukemias.

Fusion proteins contribute to leukemogenesis by an aberrant recruitment ofprotein complexes containing HDAC, histone methyl transferase (HMT), andDNMT activities, which modify the chromatin structure and silence key myeloidgenes (Moe-Behrens and Pandolfi, 2003; Bhalla, 2005; Di Croce, 2005; Melnick,2005; Minucci and Pelicci, 2006; Zardo et al., 2008).

Acetylation of specific histone aminoacidic residues is maintained by a balancebetween the activities of two principal groups of proteins, HATs and HDACs;additional data have shown that methyltransferases can also modify histoneaminoacidic residues, while HDACs can modify deacetylase transcription fac-tors or other cellular proteins (such as the cytoskeleton protein α-tubulin, nuclearimport protein importin-α7, signal transduction protein β-catenin, DNA repairenzyme KUZ 0, and heat shock protein 90) (Bhalla, 2005). Moreover, cyto-sine methylation can attract methylated DNA binding proteins and HDACs tomethylated CpGs islands during gene silencing.

All these events are able to create a complex epigenetic network that is pro-foundly altered in the pathogenesis of human leukemias, even if the precisemolecular mechanisms remain to be understood (Bhalla, 2005).

A particular characteristic of some leukemia and lymphoma is the abnormalactivity of HATs and HDACs; an interesting example is that in which the twoHATs p300 and CBP (CREB-binding protein), usually considered to be tumorsuppressors in correlation with several oncoproteins (such as p53, pRB, Myb, Jun,and Fos), are disrupted in leukemia by chromosomal translocations. Chromosomaltranslocations produce fusion proteins in which, frequently, HATs are aberrantlyfused with many partners, thus influencing profoundly epigenetic mechanisms.

9.2.1.1 MLL Translocations. Among mammalian Trithorax group (TrxG) pro-teins, the mixed-lineage leukemia (MLL) gene dysfunction is related to leukemo-genesis. The MLL product is a large multidomain protein ubiquitously expressedin hematopoietic cells including stem and progenitor populations; like othermethyltransferases, it is a part of large nuclear complexes that include manycomponents of the TFIID transcription complex (Krivtsov and Armstrong, 2007;Zardo et al., 2008).

MLL rearrangements are present in more than 70% of pediatric leukemias(less than one year of age) and in approximately 10% of AMLs in adults and intherapy-related leukemias (Zardo et al., 2008).


The association of MLL translocations with a young age at diagnosis, theirpresence in about 80% of the cases of pediatric AML and ALL, because approx-imately 50% of them are not responsive to chemotherapy treatment, and thepoor clinical outcome of patients have generated much interest in the biology ofMLL-rearranged leukemias (Stam et al., 2006; Krivtsov and Armstrong, 2007).In addition, MLL rearrangements have been detected in newborns and abortedfetuses, indicating that the disease could develop in the uterus (Klose et al., 2007).

In association with different sets of proteins, MLL positively regulates Hoxgene expression that participates in the development of multiple systems, includ-ing the hematopoietic system.

MLL binds directly to the Hox gene promoters and methylates H3K4, therebycontributing to their correct expression in normal hematopoietic cells and theirgradual downregulation during differentiation (Fig. 9.3).

Moreover, MLL recruits histone acetyltransferases CREB Binding Protein(CBP) and Males absent on the First (MOF), thereby resulting in acetylation ofhistones H3 and H4 at target genes (Jiang and Milner, 2002; Yokoyama et al.,2002).

In both ALL (in about 80% of cases) and AML, MLL proteins are fusedwith more than 60 other proteins, normally involved in acetylation, deacetyla-tion, and methylation mechanisms (such as CBP, p300, and several subunits ofthe SWI–SNF complex) (Fig. 9.4). Recent data have revealed that the translo-cation partner genes (TPGs) are more than 87 in number and even if at least 52

Normal Hox/meis

Hox/meis

Stem cell

Stem cell

Leukemia

Precursor

Precursor

rapid selfrenewal

MLL fusion

Mature cell

Mature cell

FIGURE 9.3 Normal and pathological function of Hox genes and MLL proteins: inleukemic cells, MLL fusion proteins can block Hox genes expression, thus preventing acorrect cell maturation. (From Slany, 2005.)


Xq24

1q322p37

2q37

7q22

7q328q11

8q249p11

9q33

10q2211p1511q13

MLL and partners—recurrent translocations. Editor 06/2000; last update 0ε

12p1312q13

12q2414q11

14q32

17q11

18q1218q23

20q1321q11

Xq13 (AFX1) Xq22 (Septin2)1q32 (AF1P)

1q21 (AF1q)

2q11 (LAF4)3q21 (AF3p21)

3q25 (GMPS)

3q28 (LPP)

4q21 (AF4)

4q21 (SEPT11)

5q31 (GRAF)

5q31 (AF5q31)

6q21 (AF6q21)

6q27 (AF6)

9p23 (AF9)

9q4 (AF9q34)

9q34 (FBP17)10p12 (AF10)

10p11 (AB11)11q23 (MLL duplication)

11q23 (LARG)

14q24 (gephyrin)

15q14 (AF15q14)

15q15 (AF15)

15q15 (not AF15)

16p13 (CBP)

17p13 (GAS7)

17q21 (AF17)

17q21 (RARA)17q25 (MSF)

19p13. (ELL)19p13. (EEN)

19p13.3 (ENL)

22q11 (hCD Crel)22q13 (P300)

FIGURE 9.4 MLL and partners in leukemias. (From the Atlas of Genetics and Cytoge-netics in Oncology and Haematology . URL http://AtlasGeneticsOncology.org.)

functionally diverse MLL TPGs have been described, only a few of them can beclassified into groups (Meyer et al., 2006; Krivtsov and Armstrong, 2007).

MLL translocations encode MLL fusion proteins in which the typical domainresponsible for the H3K4 methylation (SET domain) is lost, thus promotinga leukemogenic gene-expression program through more than one mechanism.One exception in which the SET domain is retained concerns the MLL-PTDtranslocation; such an example clearly indicates that many others properties offusion partners may be equally relevant in conferring leukemogenic activities toMLL fusion products (Zardo et al., 2008).

These aberrant protein complexes interfere with normal epigenetic controls,silencing or activating specific genes at inappropriate times, hence preventing acorrect cell differentiation (Ayton and Cleary, 2003; Daser and Rabbitts, 2004).As expected, aberrant MLL fusion proteins disrupt patterns of Hox genes expres-sion, thereby promoting aggressive leukemias.

In the MLL-ENL t(11;19) translocation, ENL, being a component of theSWI–SNF complex, is responsible for the upregulation of Hoxa7 and Hoxa9genes. Binding with the acetyltransferases CBP t(11;16) or p300 t(11;22) retainsthe bromodomain and HAT domain, thereby increasing histone acetylation ofgene promoters that are usually targeted by MLL (Vire et al., 2006).


In a large cohort of infant MLL-rearranged ALL (36 patients), 100% of casesstudied have the 5′ promoter region of the FHIT (fragile histidine triad) genehypermethylated.

The human FHIT gene codifies for a putative tumor suppressor with aproapoptotic function through a caspase-mediated pathway; 5-aza-2′-deoxy-cytidine treatment restores gene expression in leukemia, thus suggesting a strongcorrelation between hypermethylation and FHIT inactivation in the developmentof MLL-leukemia (Yu et al., 2004; Stam et al., 2006).

9.2.1.2 AML1 Translocations. The t(8;21) translocation results in a fusionbetween the AML1 , also known as RUNX1 or CBFA2 , gene at 21q22 and theETO (MTG8 ) gene at 8q22 (Zhang and Rowley, 2006). It is the most commontranslocation in AML and is characterized by a good response to therapy and aprolonged disease-free survival.

The AML1-ETO fusion protein has a dominant negative effect over the nor-mal AML1 gene through the aberrant recruitment of the N-Cor/Sin3/HDAC1complex directly to AML1 consensus sequences, thus keeping the chromatin ina deacetylated state and blocking myeloid differentiation (Hug and Lazar, 2004).The AML1-ETO fusion product can also physically interact with transcriptionfactors, modifying their activity and the expression of their target genes. Theexample of AML1-ETO fusion proteins shows a clear link between leukemias,fusion proteins, and aberrantly hypoacetylation mechanisms.

Recent findings indicate that AML1-ETO upregulates genes such as Jagged1 ,thus altering the Notch signaling, plakoglobin and β-catenin, and increasing theactivity of the Wnt pathway. Therefore, the expression of AML1-ETO can becorrelated with the blockage of differentiation of committed myeloid progenitorsand an increase in the self-renewal potential (Zardo et al., 2008).

9.2.1.3 PML-RAR Translocation. In about 90% of the cases of acute promye-locytic leukemia(APL), the product of the t(15;17) causes fusion between thePML gene and the all trans-RARα (PML-RARα).

PML-RARα chimera binds and inhibits genes regulated by RARα (retinoicacid receptor α), recruiting chromatin modifiers (HDACs, HMTs, DNA/histonemethyltransferases, and methyl-binding domain proteins) and, therefore, caus-ing their transcriptional silencing and blocking of hematopoietic differentiation(Fig. 9.5). More precisely, in normal cells, RARα binds to gene promoters in pro-tein complexes including HDAC and HAT activities; at the right time, retinoicacid (RA) dissociates from the corepressor complexes and binds to HATs andthe coactivator, thus maintaining chromatin in an open state and activating geneexpression. In leukemic cells, PML-RAR shows a stronger affinity for corepressorproteins and, even in the presence of high concentrations of RA, the coactivatorcannot bind and activate gene transcription.

A prominent role has also been demonstrated for RXR, the heterodimeric part-ner of RARα; in fact, the entire PML–RARα–RXR heterooligomeric complexseems essential for leukemic transformation (Zeisig et al., 2007; Zhu et al., 2007;Zardo et al., 2008).


3

PML (15q22) RARA (17q21)relative

frequency

4 5 6 3 bcr1 55%

5%

40%

3 4 5 6 3

3 3

bcr2

bcr3

FIGURE 9.5 Schematic diagram of the PML-RARα transcripts. RARA breakpointsoccur always in intron 2, while three different breakpoints on the PML gene have beendefined: intron 6 (brc1, occurring in 55% of the cases), exon 6 (brc2, 5%) and intron 3(brc3, 40%).

In addition, it has been observed that the PML-RAR chimera can interactwith p53 and, by recruiting HDACs, causes its deacetylation and sub-sequent degradation via the proteasome/MDM2 pathway. The principal effectof this interaction is an enhanced resistance to stress (Insinga et al., 2005). Italso seems that the wild-type PML is necessary for the binding between p53and PML-RAR; in line with these observations, it has been demonstrated thatPML mice null cells expressing PML-RAR do not have enhanced resistance tostress (Insinga et al., 2005). As p53 is rarely mutated in AMLs, its involvementwith PML-RAR chimera represents the first clear example of alterations of p53protein acetylation and tumorigenesis.

9.2.1.4 TEL1-AML1 Translocation. The TEL1-AML1 fusion protein, alsocalled ETV6/CBFA2, is the most typical translocation t(12;21)(p12;q22) inpediatric B-cell ALL. The TEL1-AML1 chimera seems to have a prenatalorigin, even if it develops at about two to five years of age in about 25% ofcases.

Compared to the other leukemic translocations, the TEL1-AML1 is gener-ally characterized by a more favorable prognosis (Klose et al., 2007), even ifdifferences in outcome can depend on the diverse chemotherapy regimes and


PNT(a)

(b) PNT

GAATAGCAG AATGCATAC AML1 exon 2

AML1 exon 3ATGCCAGCAGAATAGCAG

mSin3A

Dimerization

mSin3AN-CoR

RUNT

DNA binding

Transactivation

TEL nt 1033

AML1

AML1TEL

TEL

FIGURE 9.6 (a) Structure of the major forms of the TEL/AML1 fusion gene: the nt1033 represents the breakpoint of the TEL gene, thus fusing the PNT region with AMLexon 2, or more rarely, with AML exon 3. (b) The arrows indicate the binding sites forcorepressors, such as mSin3A, N-CoR, and mSin3A in TEL/AML1 fusion protein. (FromLoh and Rubnitz, 2002.)

individual host response to therapeutic treatment. Further, it seems that relapsedTEL-AML1-positive leukemia could be considered as an outgrowth of a sec-ondary leukemia that shares a common initial event with the first (Loh andRubnitz, 2002).

In normal cells, the TEL gene encodes a nuclear and modular phosphoproteinbelonging to the ETS transcription factor family (Yeh et al., 2002); in leukemias,the entire coding region of the AML1 gene is aberrantly fused with the N terminusof the TEL gene containing a PoiNTed (PNT) protein interaction domain, an helix-loop-helix (HLH) domain, and a central region, thus producing a fusion protein(Fig. 9.6a) (Loh and Rubnitz, 2002).

This aberrant chimera is able to inhibit the expression of genes normallyactivated by AML1 , such as IL3 (Uchida et al., 1999). Moreover, the fusionprotein TEL-AML1 can form homodimers or heterodimers with wild-type TELor others ETS proteins, thus working as a gene repressor (Chakrabarti et al.,2000).

Fusion proteins can interact with transcriptional corepressors, such asmSin3A, N-Cor, and HDAC3, thus resulting in the formation of multiproteiccomplexes. The histone–deacetylase activity of these complexes inducesconstitutive repression of specific genes, contributing to leukemogenesis (Vireet al., 2006) (Fig. 9.6b).

9.2.2 Aberrant Methylation in Leukemia

9.2.2.1 Cyclin, Cyclin-Dependent Kinase Inhibitors, and Other Genes. DNAmethylation of promoter-associated CpG islands is frequently observed in adult


and pediatric leukemias and it has been associated with gene silencing andmalignant transformation (Garcia-Manero et al., 2009).

Several studies have shown that leukemia is characterized by the concomitantmethylation of multiple genes and that the number of methylated genes is asso-ciated with a worse outcome. Moreover, differences in methylation patterns areobserved among adult and childhood leukemias and are finally correlated withprognostic differences (Canalli et al., 2005; Garcia-Manero et al., 2009).

Methylation profiling studies have identified a great number of genesfrequently methylated in the major subtypes of hematopoietic malignancies,such as E-cadherin , the cell cycle regulator p16, the apoptosis regulatorDAP kinase 1, RARβ2, and CRBP1 (Galm et al., 2006). At the same time,abnormal hypomethylation has been correlated to the increased expressionof important leukemia-associated oncogenes such as cyclin D2, Bcl2 , andH-Ras (Bhalla, 2005). Because the deregulation of cell cycle genes, suchas cyclins , cyclin-dependent kinases (CDK ), and their inhibitors (CDKI ),including CIP /KIP (p21WAF, p27, and p57) and INK4 (p15 and p16), isfrequently observed in tumors, many researchers have investigated the epigeneticaberrations that can change the expression of these important genes (Gutierrezet al., 2005). Early indications suggest that p14 and p16 are rarely methylatedin ALL; p57 is methylated in 50% (31 of 63 patients) of adult ALL and rarelyin pediatric ALL. Still, p73 is methylated in 20% of cases in adults (17 of80 cases) and in approximately 19% of children (3 of 16 cases) with ALL;however there is little data concerning p27, p53, and RB in leukemias. It seems,however, that there are differences also for methylation status of p53, RB ,and CIP /KIP genes between adult and childhood ALL (Gutierrez et al., 2005;Garcia-Manero et al., 2009).

The two CDK inhibitors, p16 and p15 genes, both mapped at the 9p21 region,encode proteins involved in the regulation of the RB function; more precisely,they modulate the CDK4:6 interaction with D1 cyclin, thereby inactivating RBand inhibiting cell growth (Guo et al., 2000). Recent data have shown that p16and p15 genes are altered in leukemias, not only for deletion or mutation but alsofor methylation-associated transcriptional silencing. Methylation of CpG islandsin the gene promoter seems to be the primary cause of inactivation of thesegenes in hematological malignancies (Omura-Minamisawa et al., 2000), and itis responsible for the abrogation of the RB-p16 pathway. Researchers examinedthe methylation status of the p15 and p16 genes in adult and childhood ALLand AML, showing that these two genes are frequently hypermethylated in thesecancers. In particular, p15 gene is hypermethylated in about 48% of childhoodAML patients (14 of 29 cases), while p16 is hypermethylated in about 38% ofchildhood AML patients (11 of 29 cases), thus suggesting a prominent role forthe RB/p16 pathway in the pathogenesis of this disease (Guo et al., 2000). Thep15 CDK inhibitor (p15INK4b or CDKN2B ) is considered to be a tumor sup-pressor for myeloid leukemia in mice (Jouvenot et al., 2003), and it is aberrantlymethylated in childhood and adult human leukemias (AML, ALL, and CML),but not in lymphoma and solid cancers (Koduru et al., 1995; Drexler, 1998).


Interestingly, the degree of methylation can increase during the progression ofleukemia, thereby showing p15-INK4b as a useful marker of this disease. Further-more, almost 95% of acute leukemias have the calcitonin gene hypermethylated,while, in acute lymphocytic leukemia (ALL), the methylation of the p21WAF1gene is correlated with a poor clinical outcome (Bhalla, 2005).

Independent studies have recently shown that Wnt-related genes and kinases,such as ephrin receptors and their ligands, are epigenetically inactivated in ALL(Agirre et al., 2006; Garcia-Manero et al., 2009).

The DLX genes (DLX17 ) are implicated in hematopoiesis and regulated withthe highly dynamic spatiotemporal expression. As they are characterized by largeCpG islands in their 5′ region and involved in resistance to apoptosis of severaltumor cell lines, their potential role in leukemogenesis has recently been studied(Campo Dell’Orto et al., 2007). To investigate the role of DLX genes, researchersanalyzed DLX CpG island methylation in a group of pediatric B-ALL charac-terized by MLL-AF4 and TEL-AML1 translocations and have identified, for thefirst time, frequent hypermethylation of the DLX3 gene in pediatric B-ALL char-acterized by MLL-AF4 chromosomal rearrangements (Campo Dell’Orto et al.,2007).

The Apoptosis-stimulating protein of p53 (ASPP) family of proteins (ASPP1,ASPP2, and iASPP) increase the apoptotic effect of p53 as they induce thetranscription of proapoptotic genes such as BAX and PIG3 . In wild-type TP53tumors such as leukemia where the percentage of mutations is <10%, differentmechanisms may be involved in the inability of p53 to induce apoptosis; inparticular, more authors have suggested that resistance to apoptosis can dependon alterations of ASPP1 and ASPP2 (Samuels-Lev et al., 2001; Bergamaschi etal., 2003; Slee and Lu, 2003).

In fact, more recently, analyses of 180 patients with ALL showed that theASPP1 promoter was hypermethylated in 25% of the cases, thus explaining whyresistance to apoptosis was observed in tumors with normal p53 (Agirre et al.,2006).

Abnormal expression of HOX genes is strongly correlated with the develop-ment of hematopoietic malignancies. As described previously, MLLs regulateHOX genes positively in normal hematopoietic cells, while aberrant MLL fusionproteins disrupt patterns of HOX genes expression, thus promoting aggressiveleukemias. In addition, simultaneous overexpression of multiple HOX genes isassociated with specific subtypes of AML and ALL (Armstrong et al., 2002;Debernardi et al., 2003; Strathdee et al., 2007).

Among the members of the HOX gene family, HOX4 and HOX5 were hyper-methylated in all types of leukemias examined, including childhood ALL andAML (Strathdee et al., 2007). Moreover, it seems that the abnormal expressionof both genes is correlated with clinical characteristics that identify leukemiasubtypes. In addition, unlike in adult leukemias, comethylation of the HOXgenes is positively correlated in childhood leukemias (Crooks et al., 1999; Fulleret al., 1999). Even if it is not yet clear why HOX loci are more susceptible tohypermethylation during childhood leukemogenesis, it has been suggested that


tumor-suppressor activity may regard multiple HOX genes in early life, but islimited to a small number of HOX genes in the adult (Strathdee et al., 2007).

Epigenetic silencing of the interleukin-12 receptor β2 (IL-12Rβ2) gene is animportant feature in pro-B, early pre-B, and pre-B pediatric ALL and is correlatedto tumor cell growth. In particular, the IL-12Rβ2 gene is hypermethylated in CpGislands in exon 1, and primary tumor cells obtained from pediatric pro-B, earlypre-B, and pre-B ALL (30 cases) do not express the IL-12Rβ2 chain.

As methylation of IL-12Rβ2 is not detected in normal bone marrow pro-B andpre-B cells, the authors suggest that silencing of this gene is a new mechanismof tumor escape in pediatric B-ALL (Airoldi et al., 2006).

9.3 NEUROBLASTOMA

Neuroblastoma (NB) is a PNT (peripheral neuroblastic tumor) that represents oneof the most common solid cancers in children under the age of five and accountsfor 8–10% of all pediatric cancers. This kind of tumor originates from embry-onic neural crest-derived cells. Accordingly, it shows neuroectodermal propertiesconsisting of a heterogeneous mixture of different kind of cells, such as those thatnormally constitute the sympathetic nervous system and the adrenal medulla—inparticular, nerve sympathetic cells, sympathetic ganglia, neuroendocrine chro-maffin cells, and mature ganglion cells. Arising from multipotent neural crestcells, NB often shows several cell phenotypes, and this heterogeneity is used forprognosis (Shimada et al., 1999). In fact, neuroblastic tumors have been classi-fied in different risk categories according to their cellular differentiation stage.The most aggressive tumors are undifferentiated malignant NBs (Schwannianstroma-poor) and the intermediate ones are ganglioneuroblastomas (Schwannianstroma-rich) up to the well-differentiated benign ganglioneuromas (Schwannianstroma-dominant) (Shimada et al., 1999). The extensive cell heterogeneity ofNB accounts for several cell types, among which some are pluripotent and candifferentiate into other NB cell types. In particular, three distinct cell types arepresent in NB: neuroblast (N-), stromal (S-) and intermediate (I-) types that dif-fer markedly in malignant potential, with the I-type being the most malignant,as measured by both the colony-forming efficiency in soft agar and the ability toform tumors in athymic mice (Lechner et al., 2000). I-type cells can differenti-ate into either neuroblasts or stromal cells. Most likely, NBs originate from allof these cell types or from a pluripotent precursor cells. Even if disseminated,most NBs, when present in children under one year of age, undergo matura-tion into benign ganglioneuromas or regress spontaneously. Conversely, a verypoor prognosis is related to tumors in children older than one year with highlydisseminated disease at diagnosis. In this latter case, relapse after conventionaltherapy and/or drug resistance are features of the tumor and are responsible for<15% of a three-year survival. Mortality associated with this kind of pediatrictumor accounts for 30–60% overall, being <10% for patients in the low-riskand intermediate-risk groups and more than 60% for patients with high-risk dis-ease. The amplification of the MYCN oncogene and 1p36 allelic loss represent

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the genetic alterations most frequently found in NB, both being associated with apoor prognosis. However, MYCN amplification occurs only in about 25% of NBsand is associated with advanced-stage disease, rapid tumor progression, and lowsurvival rates. Further, gains of chromosomes 4q, 6p, 7q, 11q, and 18q, amplifica-tion of MDM2 and MYCL genes and 17q trisomy, and allelic losses at 11q, 14q,and 10q have also been described (Brodeur, 2003). Finally, microsatellite insta-bility (MSI) was detected in 14% of neuroblastic tumors (Lazcoz et al., 2007).Interestingly, some molecules important in NB pathogenesis, such as MYCN andTrk, are shown to be some of the major players in embryonic development andneural crest differentiation, suggesting that their deregulation or mutation can beconsidered to be the result of a developmental defect probably occurring dur-ing the normal cell differentiation processes (van Noesel and Versteeg, 2004).Altogether, these genetic aberrations have been found in only 25–50% of neu-roblastic tumors. Thus, new molecular markers for NB prognosis are needed(Alaminos et al., 2004). By using high-throughput analysis and biotechnologicaltools, many epigenetic molecular alterations have been identified, allowing thecorrelation of many biological pathways to the pathogenesis of NBs. Moreover,profiling the status of CpG island hypermethylation in human primary NBs mayhave clinicopathologic value because clustering of gene hypermethylation mayhelp discriminate between high- and low-risk NB patients. However, a compre-hensive methylation profiling of a large series of neuroblastic tumors has to becarried out on other cohorts of primary tumors. A number of studies have beencarried out to determine the role of epigenetic disruption in the developmentof NB tumors. Even if there are some conflicting results regarding the singlegene involved, it seems that alteration of the epigenetic control of gene expres-sion contributes to tumorigenesis. In general, methylation-mediated inactivationof a subset of tumor suppressor genes (TSGs) may cooperate with genetic alter-ations including MYCN amplification. Methylation-mediated inactivation of asubset of TSGs may cause genetic changes that lead to progression of NB orgenetic alterations in advanced NBs, which may result in a CpG island methy-lator phenotype (CIMP). CIMP accounts for methylation of CpG islands both inexonic and promoter regions. It has been reported that exonic CpG islands aremore susceptible to methylation than promoter islands (Ushijima and Okochi-Takada, 2005). The canonical prognostic markers are not always sufficient togive a good estimate of the cases currently stratified into the intermediate-riskgroup. Among these cases, those without MYCN amplification show a negativeoutcome.

9.3.1 Aberrant Methylation in Neuroblastoma

It is well known that aberrant hyper- and hypomethylation can inactivate TSGsand reactivate the expression of potential oncogenes respectively. Despite thewealth of information regarding most adult tumors, only limited knowledge isavailable on the epigenetic modifications that characterize NB. Nevertheless,in the last few years, several observations have been made on the relevance of


methylation of cytosines 5′ of guanine residues (CpG) in gene-promoter regionsin this children’s neoplasm. This kind of methylation represents a regulatorymechanism during normal cell development. It is a reaction catalyzed byDNMTs that are able to convert cytosine into 5-methylcytosine. Because of itssusceptibility to deamination, this modified residue becomes a high-frequencymutation point, leading to carcinogenesis or epigenetic reprogrammingdepending on the cellular context (Morgan et al., 2004). Moreover, the aberrantmethylation of CpG-rich island (CGI) in cooperation with posttranslationalhistone modifications, such as acetylation and deacetylation, alters geneexpression by influencing chromatin condensation and the accessibility oftranscription factors to DNA. Changes in methylation profiles could derive fromnormal genomic restriction rather than development of a cancer phenotype.Nevertheless, the variability in the proportion of N-, S-, and I-type cells withinthe tumor may mask real differences in hypermethylation profiles. In addition,the “neuronal” component can differ in extent of maturation, ranging fromdifferentiated or poorly differentiated to undifferentiated cells, with a largerextent of differentiation signifying a better prognosis for the patient. Moreover,preliminary immunocytochemical studies have revealed the presence of cellsanalogous to the malignant I-type stem cells of cell lines in all tumors atvarious frequencies. Thus, NB tumors comprise multiple cell types, with eachtype theoretically having a distinct hypermethylation and a gene expressionprofile. Even with this inherent variability, methylation profiles may proveprognostic in NB and other cancers. However, care is necessary when attributingthe development of cancer to such changes (Salmaggi et al., 2004). Thisheterogeneity is likely to be a consequence of differential aberrant epigeneticpatterns at the level of genes involved in differentiation. MYCN amplification,1p36 deletion, and 17q duplication are genetic alterations that are frequentlyobserved in NB. Although MYCN amplification is considered to be the mostimportant molecular prognostic marker of high aggressive tumors, it is presentonly in 25–30% of NBs (Oppenheimer et al., 2003). Rapidly progressiveNBs often develop in the presence of the MYCN single copy. In order not tounderestimate the proportion of high-risk patients, it is necessary to identifyother predictive biomarkers that would be more representative of the disease.In this regard, many research groups are pursuing the task of characterizingmethylation profiling in order to obtain a classification of tumor subtypes withdifferent biological and clinical characteristics. Recently, it has been shown thathypermethylation is correlated to specific clinical stages; more precisely, fre-quencies of methylation in NB are lesser at stages I and II than at stages III andIV of the disease (Michalowski et al., 2008). In fact, a new concept concerningthe influence of epimutations on human tumorigenesis is emerging. On the basisof the increasing number of new data supporting the observation that the moreaggressive NB tumors have multiple genes aberrantly methylated, it is likelythat the collection of epigenetic changes at more than one tumor-associatedgene or locus acquires clinical significance. It raises the appealing possibilityof enrolling new therapeutic approaches that include demethylating agents (Ren

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et al., 2004). The study of Alaminos et al. represents an attempt to restrictneuroblastic tumors on the basis of the methylation state of 45 repre-sentative genes and to determine the association between methylation,clinical–pathological classification, and survival of patients. By usingmethylation-specific polymerase chain reaction (MSP), according to theirmethylation status, 118 primary tumors were distinguished into stroma-rich andstroma-poor tumors, the latter being further subdivided into more aggressivetypes, with metastatic spread, and localized low-risk tumors. Moreover, theanalyses of 10 homogeneous populations of S-type (stromal or substrateadherent), N-type (neuroblastic), and I-type (intermediate or stem) NB cell linesshowed that it is possible to establish a relationship between cell phenotypeand methylation profiles of the examined genes. This study provided potentialclinically relevant methylation signatures in distinct biological NB subtypes(Alaminos et al., 2004). Another study focused on assessing the methylationpattern of 11 genes in 31 NBs with respect to their benign counterparts, 13ganglioneuroma. By MSP and bisulfite restriction enzyme analysis (BRE), fivegenes were aberrantly methylated in NB: CASP8 , 14.3.3σ, �Np73, RASSF1A,and DcR2 , compared with ganglioneuroma (Banelli et al., 2005b). Moreover,these authors found specific methylation profiles at the level of these genes inbiologically and clinically distinct groups of the neuroblastic tumors, specificallyganglioneuroma and NB, and between MYCN amplified and not amplified NB.It has been reported that a methylation phenotype involving at least four genesis necessary to suggest a correlation with decreased survival in patients withNB (Yang et al., 2007). More recently, Hoebeeck et al. have confirmed theimportance of the aberrant methylation of CpG islands in NB (Hoebeeck et al.,2009). The identification of particular methylation profiles for any tumor ortumor subtype may be useful for improving the correct prediction of the diseaseoutcome. That the methylation profile of promoter CpG islands of varioustumor-related genes might be a hallmark of human tumors has been suggestedin a few reports in the past years (Teodoridis et al., 2008). Nevertheless, evenother CpG islands outside promoter regions have shown an intrinsic tendencyfor methylation according to the tumor type, sometimes not correlating with lossof gene expression. It is known that exonic CpG islands are more susceptibleto methylation than promoter islands (Ushijima and Okochi-Takada, 2005). Theextensive methylation of multiple CpG islands, independent of their influenceon gene expression, represents a specific signature for each kind of tumor that isdefined as CIMP, or CpG island methylator phenotype. Evidences from globalmethylation studies attest the association between CIMP and poor prognosis inNB, conferring a potential clinicopathologic value to CIMP (Abe et al., 2005,2007). By using these wide screening methods, some NB patients have beenclassified into either CIMP(+) or CIMP(−), according to the levels of methyla-tion of a specific group of CpG islands. The former, characterized by methylationlevels higher than 60%, had a poorer overall survival (OS) than CIMP(−) cases,whose methylation levels were lower than 40%. Unlike a single or a small groupof aberrantly methylated genes, CIMP value seems to better correlate with the


canonical parameters used to stratify NB cases in the right clinical risk groups.This can be explained by the fact that silencing of an individual gene mayaccount for the poor prognosis of only a fraction of NB cases. Such an approachhas been utilized for other tumor CIMPs (Xue Jun et al., 2003). Through agenome-wide scanning method, methylation-sensitive representational differenceanalysis (MS-RDA), Abe et al. searched for differences in CpGs methylationbetween NBs with a good and a poor prognosis. At the level of the CpG-richregions differentially methylated, MSP analysis revealed the presence of specifichypermethylation of PCDHB, PCDHA, hepatocyte growth factor-like proteinHLP, DKFZp451I127 , and cytochrome p450 CYP26C1 (CYP26C1 ) genes inNB cell lines established from NB tumors with poor prognosis. When theMSP analysis was extended to 140 Japanese primary samples, methylationof different CGI groups were found to be closely associated with each other,defining a CIMP for NB with a poor prognosis, in which multiple genes weresimultaneously methylated (Abe et al., 2005). Notably, methylation of Proto-cadherin β (PCDHB) gene family, the locus used for the sensitive detection ofthis CIMP, did not affect gene expression. The presence of the same CIMP andits clinical value were confirmed in another cohort of 152 German NB primarysamples (Abe et al., 2005). Other data are consistent with the assumptionthat aberrant methylation of multiple genes is likely to contribute to NBpathogenesis.

Currently, these research works represent exploratory efforts toward the estab-lishment of universal and standardized methods for automated analysis. Impor-tantly, to investigate the clinical relevance of the epigenetic aberrations in NB,many reports have evaluated the associations between gene methylation, estab-lished prognostic factors, and the outcome. The methylation pattern is not ran-dom in NB; instead it represents a portrait of the development and evolutionof this pediatric malignancy (as appears for the other tumors in this chapter).Methylation-associated silencing of TSGs is a frequent event in NB. It is likelyto represent a pathogenetic phenomenon considering that treatment with thedemethylating agent 5′-aza−2′-deoxycytidine (5-Aza-dC) significantly inhibitsNB growth in vivo. Altogether, these studies have highlighted several genes astargets of specific aberrant methylation in NB. As a matter of fact, genes such asMGMT (O6-methylguanine-DNA methyltransferase), PTEN, MXI1, FGFR2 , andTIMP3 have been found to be hypermethylated in neuroblastic tumors. TIMP3was hypermethylated in 51% of NB at diagnosis (45 cases) and in 59% at relapse(10 of 17 cases) (Michalowski et al., 2008). While the methylation status ofPTEN, MXI1 , and FGFR2 was found to be correlated with their lack of expres-sion, that of MGMT was not (Lazcoz et al., 2007). In NB, many conflictingresults have been obtained till now regarding the aberrant epigenetic profiling ofspecific genes. A rather common situation seen in NB is the higher frequencyof methylation observed in cell lines, compared to that in primary tumors. Thiscould be explained by taking into account the heterogeneity of the cell lines that,as a consequence of their establishment as permanent cultures, do not representthe whole spectrum of neuroblastic tumors.

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Some examples are offered by some genes. DR4 , unlike the other TRAIL(tumor necrosis factor-related apoptosis-inducing ligand)-signaling mediatorsDCR1 and DCR2 , was unmethylated both in the malignant (31 cases) andbenign (13 cases) forms of primary tumors (Banelli et al., 2005b). Previously,the same region of this gene was reported to have shown methylation in 9 NBcell lines but not in the 28 NB tumors that were analyzed (van Noesel et al.,2002). Conversely, DR4 was among the most frequently hypermethylated genein 72 (50%) of 145 tumors found by the group of Alaminos et al. (2004), and itwas found to be methylated in 79% of the 14 NB cell lines (Yang et al., 2007).This divergence might depend on differences in the reaction condition and in theprimer sequences utilized. In order to allow the comparison of the methylationprofiling provided by different laboratories, it is necessary to standardize thetechniques and the scoring systems. It is also very important to define thecorrect regulatory DNA sequences to be assayed. The first evidence on thehypermethylation of the CASP8 gene in MYCN amplified tumors regarded anintragenic sequence near the third exon of CASP8 without evident promoteractivity (Teitz et al., 2000; Banelli et al., 2002). Gonzalez-Gomez et al. studiedmethylation patterns of 11 genes previously shown to be altered in other cancersin 44 NB tumors and attempted to correlate these changes with the tumorstage. No specific pattern was evident (Gonzalez-Gomez et al., 2003a,b,c).Demethylation is much less studied than hypermethylation of target genes.Nevertheless, demethylation of specific sequences has been observed in othermalignancies (Boily et al., 2004). Some genes and their epigenetic disruptionare described more extensively below.

9.3.1.1 RASSF1A. RASSF1A (Ras-association domain family 1 isoform A),located at 3p21.3 locus, has been demonstrated to function as a TSG in mosthuman cancers. As a matter of fact, its reexpression in human cancer cell linesimpairs cell growth (Rastetter et al., 2007). Genetic aberrations, such as lossof heterozygosity (LOH) and MSI at 3p21, were detected in a small percent-age (14%) of 41 neuroblastic tumors (Lazcoz et al., 2006). Otherwise, epigeneticinactivation of RASSF1A is one of the most common molecular changes in humancancers compared to those in normal tissues.

RASSF1A is frequently hypermethylated in several tumors, such as NB, medul-loblastoma (MB), rhabdomyosarcoma (RMS), retinoblastoma, pheochromocy-toma, lung, breast, kidney cancer, head and neck squamous cell carcinomas,testicular germ cell tumors, and several others, showing a frequency of 40–55%in tumors and of 86% in the representative cell lines (van Noesel and Versteeg,2004). Several studies have reported significantly high percentages of primaryNB tumors showing RASSF1A hypermethylation. The first evidence regardingRASSF1A aberrant methylation in NB was derived from Astuti and colleagues in2001 and from Harada et al. in 2002. Both groups obtained similar percentagesof primary samples harboring this epigenetic abnormality, respectively 55% (37of 67) and 52% (14 of 27) (Astuti et al., 2001; Harada et al., 2002). In Banelli’spaper, RASSF1A was semi- or completely methylated in 83% of the NBs ana-lyzed (31) in contrast to benign ganglioneuromas (13), where the gene was not


methylated (Banelli et al., 2005a). Interestingly, in this study, hypermethylationof the RASSF1A gene was significantly prevalent in the MYCN amplified samples(64%) compared to that in MYCN -single copy tumors (18%).

Previously, it was reported that the RASSF1A promoter was hypermethylatedin 55% of NB tumors, with subsequent loss of expression (Astuti et al., 2001).However, changes in the methylation profile did not correlate with the clinicalstage (Astuti et al., 2004a). Recently, Lazcoz et al. showed RASSF1A hyperme-thylation in 83% of neuroblastic tumors (29 of 35) and in 100% of the 12 NBcell lines that were analyzed (Lazcoz et al., 2006). Similarly, RASSF1A appearedaberrantly methylated in 90% of a cohort of 70 primary NB tumors, remain-ing unmethylated in the ganglioneuromas and adrenal tissue. No association wasfound between the high-risk factors and the poor outcome (Yang et al., 2007).Another recent study on 62 NB tumors reports hypermethylation of RASSF1Ain 93% of tumors at diagnosis (45 cases) and in 100% of tumors at relapse (17cases) (Michalowski et al., 2008). More recently, 71% of 42 primary tumorsshowed RASSF1A hypermethylation (Hoebeeck et al., 2009). Altogether, thesestudies illustrate the diagnostic and prognostic potential of RASSF1A methyla-tion, making it an attractive biomarker for early cancer detection (Hesson et al.,2007).

9.3.1.2 BLU (ZMYND10) and NORE1A. BLU and NORE1A, together withRASSF1A, are members of the RAS-association domain family that are frequentlyinactivated in a variety of human cancers (Agathanggelou et al., 2003). Bothgenes were found to be hypermethylated with high percentages in NB cell lines(66% for BLU and 50% for NORE1A of 12 cell lines) but not in neuroblasticprimary tumors (8% for BLU and 3% for NORE1A of 35 tumors) (Lazcoz et al.,2006). Nevertheless, other studies report high methylation frequencies even inprimary tumors. BLU is considered to be a candidate TSG that is encoded bya gene-rich deleted region in 3p21.3. Recently, the involvement of epigeneticdefects for the BLU gene in NB tumorigenesis has been highlighted by theanalysis of 62 primary tumors, out of which 17 established at relapse. The resultsreported hypermethylation of BLU in 34% of primary NB tumors at diagnosisand in 24% at relapse (Michalowski et al., 2008). Another study showed a 54%frequency of methylation for this gene in 70 primary tumors and a much higherfrequency, 93%, in a panel of 14 NB cell lines (Yang et al., 2007). There aremany other examples of genes whose frequency of methylation is higher in therepresentative cell lines than in tumor samples.

9.3.1.3 CASP8. CASP8 loss of expression has been described as a frequentevent in most of the NB cell lines and primary tumors. One possible mechanismto induce CASP8 loss of expression is the allelic loss (LOH) of human chro-mosomal band 2q33. Besides LOH, epigenetic alterations may account for TSGinactivation. In this regard, given the scarce rate of genetic mutations of CASP8in NB tumors, several investigations aimed to assess whether this gene might besubjected to epigenetic silencing. CASP8 was first reported to be methylated in

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NB by Teitz et al. with a percentage of 62% on a cohort of 42 primary tumors(Teitz et al., 2000). Interestingly, the methylated region did not result in a typicalCpG island [C + G content less than 60% (49%) and CpG/Gpc ratio below0.6 (0.25)], and the proposed regulatory region maps at the boundary betweenexon 3 and intron 3 and does not seem to exert promoter function (Banelli et al.,2002). This evidence implicates that CASP8 expression is not directly dependenton promoter methylation (Banelli et al., 2002), as shown for other genes whosesilencing correlated with poor outcome, such as PCDHB (Abe et al., 2005).

More recently, CASP8 methylation was detected in 56% of 70 NB primarytumors (Yang et al., 2007); in 38% of NB at diagnosis and in 35% of NB atrelapse (Michalowski et al., 2008); in 60% of neuroblastic primary tumors;and in 92% of cell lines (Lazcoz et al., 2006). Evidences supporting thepossibility that lack of expression of CASP8 depends on hypermethylationderives from the observation that by treating NB cells with the demethylatingagent 5-aza-2′deoxycytidine (5-AZA), it was possible to induce the cysteineprotease reexpression. Nevertheless, in another study, where 30 clinical tumorsamples of all stages were assessed for the methylation state of the regulatoryregion at the boundary between CASP8 exon 3 and intron 3, no significantcorrelation was found between CASP8 protein levels and its gene methylation(Muscat et al., 2006). The reasons for the discordant results are likely due tothe diversity of the tumor sample cohorts and the methodology used. The lossof CASP8 makes tumor cells resistant to apoptosis by inducing stimuli thatactivate TNF (tumor necrosis factor) and FAS receptors, leading to a gain in thesurvival rate. Therefore, it is not surprising that treatment with 5-AZA restoresthe responsiveness to TRAIL and FasL and induces apoptosis (Teitz et al., 2000;van Noesel et al., 2002; van Noesel and Versteeg, 2004; Fulda et al., 2006). Bystudying TRAIL-associated genes in 15 human NB cell lines, it was found thatsix genes associated with TRAIL sensitivity (CASP8, FLIP, DcR1, DcR2, DR4 ,and DR5 ) were hypermethylated in most of the cell lines.

Epigenetic repression of CASP8 has mainly been observed in neuroendocrinetumors with MYC overexpression (NB, MB, and neuroendocrine lung tumors)(Eggert et al., 2000; Mazumder and Almasan, 2002). Previous reports have shownthat CASP8 loss of expression occurs in 25–35% high-risk tumors and that it isassociated with MYCN amplification, although a direct influence of MYCN onCASP8 has not been shown (Teitz et al., 2000; Eggert et al., 2001). Neverthe-less, Fulda’s group reported no correlation between CASP8 expression and eitherMYCN amplification or aggressiveness in NB from a large cohort of NB patients(162 tissues). As a matter of fact, ectopic expression of MYCN or antisense-mediated downregulation of MYCN had no effect on CASP8 expression in NBcell lines. Even if loss of CASP8 protein expression was reported in 75% of 162NB tissues, it was not restricted to advanced stages of disease and to other vari-ables of high-risk disease, such as chromosomal aberrations, age at diagnosis, ortumor histology (Fulda et al., 2006). The fact that epigenetic aberration of CASP8is related or unrelated to high-risk prognostic markers, such as MYCN amplifica-tion, or to survival has still not been understood. In fact, new data from Hoebeeck


et al. demonstrated a significant correlation between CASP8 methylation andMYCN amplification in 115 NB tumors (Hoebeeck et al., 2009). They reportedthat 56% of 42 primary tumors showed CASP8 hypermethylation by usingmethylation-specific PCR (MSP) (Hoebeeck et al., 2009). Interestingly, recentfindings demonstrate a statistically significant association between hypermethy-lation of RASSF1A and hypermethylation of CASP8 in NB (Lazcoz et al., 2006).

9.3.1.4 HIC1. The protein encoded by the HIC1 gene suppresses cell growth inresponse to activated Ras. HIC1 was found to be methylated in 86% of NB celllines (n = 14) and in 99% of a cohort of 70 primary tumors, being methylatedeven in ganglioneuromas and adrenal tissues (Yang et al., 2007). HIC1 representsone of the rare cases in which the methylation frequency is higher in primarytumors than in cell lines. Even in a previous study, a high percentage of primaryNB tumors (43%, 12 of 28) showed hypermethylation of the HIC1 promoter byMSP (Rathi et al., 2003).

9.3.1.5 HIN1. Although the role of HIN1 as a TSG is still unclear in NB, HIN1has been shown to inhibit cell cycle reentry, suppress migration and invasion,and induce apoptosis in breast cancer cell lines (Krop et al., 2005). Its aber-rant methylation has been reported in other human cancers, including pediatricmalignancies. On studying the methylation pattern in a large cohort of tumorsamples, frequencies of HIN1 promoter methylation were found to be lower(4–12%) in NB than in other pediatric cancers, such as retinoblastomas (90%),Wilms tumors (WTs) (73%), RMSs (61%), and in adult cancers, such as breast(57%) and prostate cancers (52%) (Shigematsu et al., 2005). In a more recentstudy, HIN1 has been found to be methylated in a subset of NB tumors (21%),while being unmethylated in benign ganglioneuromas and normal adrenal tis-sues (Yang et al., 2007). At a clinical level, patients with methylation of HIN1showed a significantly reduced overall survival (OS) compared to those bearingthe unmethylated gene counterpart. Even for this gene, a higher percentage (71%)of cell lines (n = 14) was found to bear HIN1 -aberrant methylation with respectto that reported for primary tumors (Yang et al., 2007).

9.3.1.6 DCR1 and DCR2. These genes belong to the tumor necrosis factorreceptor superfamily (TNFRSF), which is responsible for cell sensitivity toTRAIL-induced apoptosis. DCR1 and DCR2 encode for two receptors that areaberrantly hypermethylated in rapidly progressive tumors (van Noesel et al.,2002). DCR2 was found to be methylated in 13 of 31 (42%) NB tumors (Banelliet al., 2005b), and in 44% of a cohort of 70 primary tumors (Yang et al., 2007).More recently, DCR1 and DCR2 were found methylated in 11% and 25% ofNB at diagnosis (45 cases) and in 18% and 29% of samples at relapse (17 cases)respectively (Michalowski et al., 2008). DCR2 methylation was not found inall the ganglioneuromas evaluated, without any statistical significance betweenMYCN amplified and nonamplified NBs. Moreover, the OS was significantlylower for patients presenting DCR2 methylation compared to those that were

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unmethylated (Banelli et al., 2005a). Partial or complete DCR2 methylationcorrelated with lower OS than the unmethylation status of the gene (Banelliet al., 2005b). It was found to be methylated in 100% of the 14 NB cell lines(Yang et al., 2007).

9.3.1.7 p73 and �Np73. The p73 is a homolog of the p53 TSG. p73 utilizestwo CpG islands containing independent promoters to code for pro- or antiapop-totic proteins: TAp73 and �Np73 respectively. The transactivating variant TAp73induces apoptosis and inhibits cell proliferation, whereas the N-terminal-deletedisoform �Np73 has the opposite function (Melino et al., 2002). �Np73 is oftenoverexpressed in highly aggressive NB independently from MYCN amplification,age, or stage (Casciano et al., 2002). The oncogenic �Np73 variant is expressedin NB in association with the demethylation of an internal promoter. Conversely,�Np73 is always completely methylated in the benign ganglioneuroma at thelevel of the internal promoter (Casciano et al., 2002). These results were con-firmed from another study on a larger group of NBs (31) and ganglioneuromas(13) (Banelli et al., 2005a). TAp73 is silenced by methylation in hematopoieticmalignancies, such as leukemia, but not in NB (Banelli et al., 2000; Tonini andRomani, 2003). This was confirmed by Hoebeeck et al. for 33 NB cell lines and42 primary tumors (Hoebeeck et al., 2009).

9.3.1.8 E-Cadherin (CDH1). The cell–cell adhesion molecule E-cadherin(CDH1 ) is abnormally expressed in NBs, a condition that has been correlatedto cancer invasion and metastasis. Aberrant methylation of the CDH1 promoterwas observed in many adult cancers but not frequently in pediatric tumors.In fact, CDH1 hypermethylation has been found in 79% of 14 NB cell lines(Yang et al., 2007) and in 43% of 11 NB cell lines even if only in 6% of the27 primary NB (Harada et al., 2002). A similar low percentage (8%) of CDH1hypermethylation in primary tumors has been recently reported by another studyon 42 NBs (Hoebeeck et al., 2009). Even if demethylation was seen in someregions of the promoter of E-cadherin in 31 cases of NBs, it was not enoughto justify the abnormal overexpression of E-cadherin in NBs (Wu et al., 2007).CDH1 hypermethylation was detected in 8% of the 42 primary NB samples,and it has been observed that there is an association between CASP8 and CDH1hypermethylation and poor event-free survival (Hoebeeck et al., 2009).

9.3.1.9 p16INK4a (CDKN2A) and p14ARF. The INK4a/ARF locus maps at9p21–22 and encodes two structurally distinct gene products, the CDK inhibitorgenes p16INK4a and p14ARF . Both the genes exert active roles in inhibitingcell proliferation. The chromosomal region encompassing the p16INK4a gene isa hotspot of LOH in NB. Nevertheless, homozygous deletions and mutations arenot involved in the inactivation mechanism of the p16INK4a gene in Brazilian NBpatients (Bassi et al., 2004). Because the expression of these genes is altered inthis pediatric cancer, other mechanisms of inactivation, such as promoter methy-lation, are likely to be involved. Although p16INK4a is a target for aberrant


promoter methylation in human cancer, the role of its epigenetic inactivation isstill controversial in NB. A few, and sometimes conflicting, results were collectedon the methylation status of this gene. What has been put on evidence is that treat-ment of p16-null NB cell lines with the DNA-methylase inhibitor 5-azacytidine(5-AZA) restores the expression of the gene (Tonini and Romani, 2003). Thepercentage of p16INK4a methylation was estimated to be 19% of a great cohortof neuroblastic tumors (n = 145) (Alaminos et al., 2004). Low p14ARF mRNAexpression has been reported in 2 out of 16 (12.5%) advanced NB tumors (stagesIII and IV) (Obana et al., 2003) and 1 out of 18 (5%) unfavorable NB tumors(stages C and D based on Pediatric Oncology Group staging criteria) (Omura-Minamisawa et al., 2000). Partial p14ARF methylation was observed in 2 outof 23 (9%) NB cell lines, GIMEN and PER-108, and this epigenetic change isassociated with transcriptional silencing of p14ARF in GIMEN cells but not inPER-108 cells by treatment with the demethylating agent 5-aza-CdR (Wang etal., 2006). In this study, methylation of the p14ARF gene promoter was observedindependently of p16INK4a methylation. Nevertheless, no hypermethylation wasdetected for both genes in 45 NB tumors in a more recent study (Michalowskiet al., 2008).

9.3.1.10 PHOX2B. The paired-like homeobox 2B (PHOX2B ) gene, at chromo-some 4p12, was found to be involved in dysautonomic disorders including con-genital central hypoventilation syndrome (CCHS), Hirschsprung disease (HSCR),and NB. A conserved homeotic transcription factor has been shown to be a reg-ulator of the development of the nervous system by promoting differentiation ofsympathetic neuroblast precursors (Pattyn et al., 1999). Interestingly, aberrationsin the p53/MDM2/p14(ARF) pathway have been described in the two NB celllines where PHOX2B was found to be methylated (Wang et al., 2006); BesidesLOH in about 10% of 45 NB tumors and no PHOX2B mutations, aberrant CpGdinucleotide methylation of the PHOX2B promoter region was found in 4 out of31 tumors and in 12.9% of 13 cell lines (de Pontual et al., 2007). These findingssuggest that the aberrant methylation of the PHOX2B promoter seems to be analternative mechanism that is as frequent as mutation in sporadic NB cases.

9.3.1.11 GABAA Receptor Genes. GABA(A) receptor subunit (GABR)genes—GABRB3, GABRA5 , and GABRG3 —are located in the human chro-mosome 15q11–13. Deletion or duplication of 15q11–13 GABR genes occursin multiple human neurodevelopmental disorders. All the three GABR genesare not imprinted in the normal human cortex, therefore they are biallelicallyexpressed in control brain samples. In NB cells, instead, GABRB3 is methylatedat certain CpG sites, leading to an epigenetic control of these genes (Hogartet al., 2007).

9.3.1.12 SEMA3B. SEMA3B (semaphorine 3B) gene maps at chromosome 3p,a location frequently subjected to large-scale LOH and encompassing numerousTSG in human NB. SEMA3B is involved in the development of normal sym-pathetic neurons, showing higher levels of expression in differentiated tumors

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with a favorable histopathology than in tumors with unfavorable histology. Infact, SEMA3B was upregulated in the SK-N-BE NB cell line following induc-tion of differentiation with RA, a known inducer of neuronal differentiation.SEMA3B epigenetic regulation was hypothesized after the observation that itsexpression was restored in NB cell lines treated with the demethylating agent5-aza-2-deoxycytidine. As a matter of fact, a high percentage of methylated CpGsites in the SEMA3B promoter was detected in tumors. Most interestingly, thefrequency of methylation of these CpG islands was about 95% in tumors exhibit-ing 3p loss, and 52% in those without loss, suggesting a reciprocal influencebetween genetic aberrations and epigenetic defects (Nair et al., 2007).

9.3.1.13 Prostaglandin E Receptor 2 (PTGER2). Prostaglandin E receptor 2(PTGER2 ) is a gene silenced by epigenetic mechanisms in advanced types of NB.Specifically, PTGER2 can be silenced by DNA methylation at the level of CpGisland by histone H3 and H4 deacetylation and by histone H3 lysine 9 methylationwithin the putative promoter region of the gene (Sugino et al., 2007). As PTGER2induces growth inhibition and apoptosis during neuroblastomagenesis, disruptionof PTGER2 responsiveness through epigenetic silencing makes NB cells moremalignant.

9.3.1.14 GTL2/DLK1. GTL2 promoter DMR (differentially methylated region)was hypermethylated in 5 (25%) of 20 NB tumors (Astuti et al., 2005). Accord-ingly, a low level of GTL2 expression was detected in these tumors. Treatmentwith 5-AzaC of NB cell lines restored GTL2 expression, indicating that hyper-methylation of this gene is associated with its silencing. Concomitantly withGTL2 hypermethylation, DLK1 expression was found to be augmented, exclud-ing the involvement of an LOI mechanism because its expression continued tobe monoallelic, even pre- and post 5-AzaC treatment (Astuti et al., 2005).

9.3.1.15 EMP3. The epithelial membrane protein 3 (EMP3 ) is a gene located at19q13.3, a site belonging to the commonly deleted 19q13 chromosomal region inNBs. This gene encodes a myelin-related four-transmembrane protein involvedin cell–cell interaction and proliferation. By means of a microarray analysis,EMP3 was the only CpG island-containing gene at the 19q13 region to be sig-nificantly downregulated in a cohort of 89 NB tumors, relative to benign tumorganglioneuromas (Alaminos et al., 2005). These findings confer a putative roleof a TSG to EMP3 . The authors, indeed, showed that restoring the expression ofEMP3 in NB cell lines resulted in reduced colony formation and tumor growthin xenografts. The fact that the absence of the transcript was due to methylationsilencing was ascertained by treating these cells with the demethylating agent5-aza-2′-deoxycytidine, thereby verifying the gene reactivation. Finally, EMP3hypermethylation was analyzed in a large group of 116 NBs, being detectedin 24% of the cases. Most importantly, Alaminos et al. established a signif-icant association between the hypermethylation status of EMP3 and the pooroutcome of the patients. Specifically, EMP3 was hypermethylated in 53.6% of


deceased cases versus 31.8% of cases when the gene was unmethylated. A sim-ilar trend was observed when the cases were stratified by stage. The mortalityfor local, regional, and metastatic stage IV NBs accounted for 33.3% and 78.6%respectively in patients with hypermethylated EMP3 , while it was 13.3% and46.1% when the gene was unmethylated. These observations confer a potentialprognostic clinical value to the EMP3 methylation status (Alaminos et al., 2005).

9.3.1.16 CD44. This gene mapping at 11p encodes for an integral glycopro-tein involved in cell–cell and cell–matrix interactions. Its overexpression hasbeen correlated with tumor progression in adult cancers. In NB, the loss ofCD44 expression associates with increased malignancy. Even if the CD44 genepromoter and exon 1 regions were found to be hypermethylated in almost allthe CD44-negative cell lines, in contrast, no CD44 gene hypermethylation wasdetected in 21 NB clinical samples (Yan et al., 2003). These evidences raisethe possibility that methylation at different sites of CD44 or other mechanismsmay control the expression of CD44 in primary NB tumors and cell lines. Amore recent study, instead, reports that 69% of 42 NB tumors showed CD44hypermethylation (Hoebeeck et al., 2009).

9.3.1.17 NR1/2. The NR1I2 gene encodes a nuclear receptor that controls theexpression of drug metabolizing and clearance molecules. It seems that the induc-tion of ectopic NR1I2 inhibited growth of NB cells, similar to other nonsteroidalnuclear receptors, such as all-trans RAR and vitamin D3 receptor. A CpG islandlocated around exon 3 of NR1I2 showed promoter activity, and its methylationstatus was clearly and inversely correlated with NR1I2 expression status. Theuse of COBRA (combined bisulfite and restriction analysis— bacterial artificialchromosome array-based methylated CpG island amplification, a method thatallows scanning of human genomes for methylated DNA sequences) on 51 sur-gically resected primary NBs showed that 9 of them (17%) showed methylationat the NR1I2 CpG islands (Misawa et al., 2005).

9.3.1.18 CCND2, TIG1, PTEN, ZMYND10, HOXA9, MDR1, TMS1, and14.3.3σ. The cell cycle regulator cyclin-D2 (CCND2 ) was reported to havereduced expression in NB. It was hypermethylated in stage IV but not instage IVS tumors (Alaminos et al., 2004). TIG1 showed a 23% methylationover a cohort of 70 primary NB tumors and a 79% methylation in 14 NBcell lines, being unmethylated in nonmalignant ganglioneuromas (Yang et al.,2007). PTEN and ZMYND10 were found hypermethylated in 25% and 15%of 42 primary tumors, respectively (Hoebeeck et al., 2009). A discordantstudy on 14 NB cell lines reports no methylation of PTEN gene (Yang et al.,2007). Promoter hypermethylation of the developmental gene HOXA9 wasdetected in 39% of 145 neuroblastic tumors (Alaminos et al., 2004). HOXA9methylation was associated with mortality in noninfant patients and in tumorslacking MYCN amplification (Alaminos et al., 2004). A correlation betweenthe hypermethylated status of MDR1 promoter and the progression of tumor

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from stage I to stage IV was identified in NB biopsy specimens (Qiu et al.,2007). Hypermethylation of the proapoptotic gene TMS1 was associated withstage IV progressing tumors, but the gene was not methylated in stage IVStumors, which undergo spontaneous regression (Alaminos et al., 2004). TMS1was found methylated in 46% of 70 primary NB tumors and in 93% of 14 celllines, whereas ganglioneuromas and adrenal control tissue showed an absenceof methylation (Yang et al., 2007). 14.3.3σ is a regulator of the cell cycleG2 checkpoint, and it is epigenetically silenced in some tumors (Fergusonet al., 2000). While it is always hemimethylated in all ganglioneuroma (benign,well-differentiated tumors), it appeared to be completely methylated in most ofthe NBs: 78% of MYCN amplified tumors against 23% of MYCN single-copyNBs (Banelli et al., 2005a).

9.3.2 Genes Unmethylated or Methylated at Low Frequency

A series of genes analyzed for their methylation state were prevalently unmethy-lated either in NB cell lines or in primary tumors. Among these, RB1 , p27,Dkk3, VHL, MCT1, PTEN , and BRCA1 showed absence of methylation, whereasENDRB, MGMT, CDH13 , and IRF7 genes were methylated in 1–30% of 14cell lines (Yang et al., 2007). As mentioned above, the p73 gene was reportedto be unmethylated in more than one independent studies (Banelli et al., 2000;Tonini and Romani, 2003; Hoebeeck et al., 2009). PRDM2 was unmethylatedon a cohort of 42 primary tumors (Hoebeeck et al., 2009). The ROBO1 geneis located on 3p12 and encodes an axon guidance receptor, a member of theNeural-CAM (NCAM) cell adhesion family receptors. Even if it was reportedto be aberrantly methylated in human cancers, ROBO1 was methylated only in3% of 33 NB cell lines, and no methylation was detected in 42 primary tumorsamples (Hoebeeck et al., 2009). Similarly, the DCC gene (18q21), involved inneuronal development, showed low methylation frequency (7%) in the same setof 42 tumors (Hoebeeck et al., 2009).

9.3.2.1 RARβ2. Treatment of NB with RA prompts growth arrest and neu-ronal differentiation of tumor cells. For these effects, retinoids have been usedin clinical trials in children with advanced NB. Among the RA target genes,the RARβ is a RA-inducible tumor suppressor that is involved in differentia-tion. Its lack of expression was associated with advanced-stage NB (Banelli etal., 2005b; Cheung et al., 1998). RARβ2 is inactivated by hypermethylation inadult human tumors (Ehrlich et al., 2002). Strikingly, the RARβ2 promoter wasfound to be prevalently unmethylated in neuroblastic tumors even under differentexperimental conditions (Harada et al., 2002; Yang et al., 2004b; Banelli et al.,2005a, 2005b). In another survey, RARβ2 methylation accounted only for 6–8%in NB tumors (10 of 145 cases), instead being more methylated in the benign gan-glioneuroma counterpart (Alaminos et al., 2004). This indicates the low frequencyof RARβ2 methylation as a peculiar epigenetic mark of neuroblastic pediatricneoplasia.


9.3.2.2 Hsp47. The gene encoding the collagen-specific molecular chaperonheat shock protein 47 (Hsp47 ) was shown to have the highest level of differentialexpression (being upregulated more than 80-fold) after 5-Aza-dC treatment in agenomewide gene expression analysis of control and treated NB cell lines. MSPand bisulfite sequencing revealed that the promoter region of the Hsp47 genewas methylated in a subset of NB cell lines (6 of 8) and primary tumors (4 of7), and the methylation status was found to be inversely associated with geneexpression (Yang et al., 2004a).

9.3.2.3 TSP1. Angiogenesis plays a critical role in the regulation of NBgrowth, and administration of antiangiogenesis agents effectively inhibits NBtumor growth in vivo (Davidoff and Hill, 2001). The angiogenesis inhibitorthrombospondin-1 (TSP1 ) was found to be inactivated by methylation insome adult tumors. The group of Yang and colleagues indicated that thetranscriptional silencing of TSP1 was caused by methylation. They found thatTSP1 was silenced in a subset of undifferentiated, advanced-stage tumors andNB cell lines. TSP1 loss of expression was ascribed to aberrant 5′CpG islandmethylation of the gene promoter because treatment with the demethylatingagent 5-Aza-dC restored TSP1 transcription in TSP1-negative NB cell lines.Furthermore, disrupting methylation with 5-Aza-dC resulted in inhibited tumorgrowth in vivo, and TSP1 expression was restored in a subset of NB xenografts.In a large portion of NB tumors (37% of 60 cases), TSP1 was methylated,irrespective of five samples of benign ganglioneuromas, of which only oneshowed methylation. Nevertheless, no correlation was reported between themethylation status of the promoter and TSP1 protein expression in 12 NB cases.The authors justified this lack of association considering that only a subset ofcells expresses TSP1 in accordance with the heterogeneous nature of NB tumortissues (Yang et al., 2003).

9.3.3 Aberrant Acetylation in NB

Little is known about the aberrant epigenetic acetylation/deacetylation dysfunc-tions occurring in NBs. One study regarding the effects of the histone deacetylaseinhibitor (HDACi) VPA on Notch signaling attests the presence of an aberrantacetylation profiling in human NB. High HDAC1 mRNA expression was asso-ciated with multidrug resistance in NB cell lines. In fact, inhibition of HDAC1expression or activity enhanced the cytotoxicity of chemotherapeutic drugs inmultidrug-resistant NB cell lines (Keshelava et al., 2007).

9.3.3.1 RARβ. HDAC inhibitors (HDACis) seem to potentiate the effects of RAin arresting NB growth, making the combination of these molecules an attractivetherapeutic approach to treat neuroblastic tumors. In an NB cell line, it was shownthat HDACis cooperate with RA to increase RARβ mRNA levels by activatingthe RARβ2 promoter that was earlier refractory to acetylation. The TSG RARβ2reexpression by HDACis might in turn modulate sensitivity to the retinoids inNB cells (de Los Santos et al., 2007b).

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9.3.3.2 p21WAF1 and p27. The effects of the HDACis TSA, sodium butyrate,and suberoylanilide hydroxamic acid (SAHA), alone and in combination withRA, cause sustained increase in histone H3 acetylation, leading to extensiveapoptotic NB cell death. Among genes targeted by this hyperacetylation in sur-viving cells, the cyclin kinase inhibitors (CKI) p21WAF1 and p27 have beenidentified. These results indicate that in a tumor context the chromatin regionsregulating the CKI p21WAF1 and p27 expressions are aberrantly deacetylated andtopologically inaccessible to the transcription machinery. Induction of p21Waf1and p27 by HDACis was further enhanced in the presence of RA (de los Santoset al., 2007a). Other studies regarding the utilization of HDACis to induce a ther-apeutic manipulation of the cancer epigenome show promising results regardingthe efficacy of these drugs (Furchert et al., 2007).

9.4 RHABDOMYOSARCOMA (RMS)

RMS is a highly malignant pediatric tumor derived from mesenchymal cells thatare committed to become skeletal muscle cells but that undergo aberrant differ-entiation (Pappo, 1995; Jarrard et al., 1999). RMS may be classified into twomajor types, the embryonal RMS (ERMS) and the alveolar RMS (ARMS). Theembryonal and alveolar types of RMS differ in histology, genetic markers, currenttreatment protocols, and prognosis. Like many other pediatric cancers, RMSs arecharacterized by typical genetic aberrations. In particular, most ARMSs containone of two recurring translocations, namely, the common t(2;13)(q35;q14) or therare t(1;13)(p36;q14). Both translocations disrupt the FKHR gene, which encodesa widely expressed transcription factor. The t(2;13) fuses part of the PAX3 tran-scription factor gene to FKHR, leading to the PAX3-FKHR chimeric protein,whereas the t(1;13) creates a PAX7-FKHR fusion. The chimeric PAX3-FKHRand PAX7-FKHR proteins are thought to alter transcription and promote tumori-genesis. Clinically, tumors expressing PAX7-FKHR are associated with favorablefeatures, and the prognosis for patients with these tumors is better than that forpatients with PAX3-FKHR-positive tumors. ARMS usually occurs on the extrem-ities of adolescents and generally has a poor prognosis. ERMS generally occursin the head and neck areas or genitourinary tract of younger children. Besides thecontradistinctive genetic aberrations, epigenetic dysfunctions that seem to exertcausative or cooperative roles in the tumorigenic development have been iden-tified in RMS. As histological subcharacterization and immunohistochemistryare not sufficient means to guarantee an appropriate diagnosis of RMS, analysisof DNA hypermethylation has become an efficient approach for the detectionof disease-associated genes as molecular markers with prognostic significancein cancers. RMS shows poor response to chemotherapy. Patients older than 10years with either ERMS or ARMS (from infancy to the age of 20) had a 5-yearsurvival rate of approximately 30%, the ARMS being the most aggressive form(Raney et al., 2001). In fact, despite advances in therapy, ARMS have a five-year survival of <30% (Linardic et al., 2007). This poor survival rate in high-riskpatients raises the necessity to investigate new classes of therapeutic agents.


In order to identify an effective therapeutic strategy, many research groupshave been testing the possibility of treating this kind of tumor with a combi-nation of agents that inhibit DNA methylation and promote histone acetylation.This procedure may lead to efficient reactivation of growth-inhibitory genes. Amultidisciplinary approach for this treatment has resulted in improvement in out-comes (Yeager et al., 2003). HDACis, such as hydroxamic acids (e.g., trichostatinA, SAHA, pyroxamide, CBHA), short-chain fatty acids (e.g., butyrates), cyclicpeptides (e.g., depsipeptide), and benzamides (e.g., MS-27-275) have been shownto exert growth-suppressive activity in a variety of tumor models (Kutko et al.,2003). Some of them are currently under investigation in phase I and phase IIclinical trials for the treatment of solid and hematological tumors in adults (Kellyet al., 2002, 2003). Kutko and coworkers showed a potential role for two HDACisin the treatment of RMS. They observed that the RMS cell lines, RD (embry-onal cell line) and RH30 (alveolar cell line), were sensitive to the effects of theHDACis, SAHA and pyroxamide (Kutko et al., 2003). In particular, SAHA andpyroxamide induced death in RMS cells in a dose-dependent manner at micro-molar concentrations, according to an increase in the level of acetylated corehistones and of the induction of p21WAF1 expression, a gene always inducedby treatment with HDACis both in vitro and in vivo (Xiao et al., 1999). It isassociated with LOH at the 11p15 locus, as well as RAS mutations (Kutko et al.,2003). Nevertheless, in many cases, it has been shown that some of the primarylesions appear not to be genetic alterations, but rather changes in methylation.

In the past years, several genes have been found to be aberrantly hypermethy-lated in RMS. Among these, there are PAX3, p21WAF1, FGFR1, MGMT, CASP8,MyoD, HIC1, HIN1, RB1, CDX1, Plakoglobin (γ-catenin), and TSSC5 . Besidesthese genes, an important role is exerted by the IGF2 and H19 imprinted genesin RMS tumorigenesis.

9.4.1 Aberrant Methylation in Rhabdomyosarcoma

9.4.1.1 PAX3. Genetic and epigenetic regulation processes are very stronglyinterconnected phenomena of gene expression. One of the first evidence regard-ing this cross-talk in human RMS is offered by the identification of aberrantmethylation of the upstream CpG island of the PAX3 gene. As mentioned above,PAX3 is one of the transcription factors that constitute PAX3-FKHR chimeric pro-tein resulting from the t(2;13) typical translocation. Methylation of PAX3 can beused for the development of an epigenetic profile for the diagnosis of RMS. Thecomparison of the methylation status of a PAX3 5′-CpG island in RMS subtypesand in normal fetal skeletal muscle showed that this CpG island is hyperme-thylated in most of ERMS examined (13 of 15 tumors), whereas most ARMS(9 of 12 tumors) and all normal muscle samples showed relative hypomethy-lation (both 18% mean methylation) (Kurmasheva et al., 2005). Further studieshave highlighted that PAX3-FKHR expression promoted both fetal and post-natal primary human skeletal muscle cell precursors to bypass the senescencegrowth arrest checkpoint in association to the epigenetic DNA methylation of


the p16/INK4a promoter and correspondingly to its loss of expression (Linardicet al., 2007). Recently a link between PAX3 and the epigenetic control of chro-matin condensation has also been identified. It has been reported, in fact, thatPAX3 is able to specifically interact with KAP1, a transcriptional corepressor,and with HP1 to facilitate the formation of a closed chromatin through histonedeacetylation and methylation. KAP1 and HP1γ compete for binding with PAX3on target promoters, resulting in a repressive transcriptional phenotype (Hsiehet al., 2006).

9.4.1.2 p21WAF1. p21WAF1 suppression seems to provide an advantage forRMS cell proliferation, as the restoration of the gene in those cell lines withabnormally low levels of p21WAF1 leads to growth arrest (Knudsen et al., 1998).p21WAF1 was found to be hypermethylated at one of the internal CpG islands ofthe promoter region, at the level of the Sis-inducible element (SIE1), a responsiveelement of STAT transcription factors. Specifically, 13 of 26 (50%) primary RMStumors and 2 of 5 cell lines have been reported to show methylation at this site.Moreover, this epigenetic feature was responsible for p21WAF1 silencing and forthe inability of STAT1 protein to bind to SIE1 in response to interferon-γ (IFN-γ).Indeed, 5-AZA restored both the p21WAF1 expression and the responsivenessto IFN-γ (Chen et al., 2000). This is an example of how such an epigeneticabnormality may influence the complex pathway involved in the responsivenessof cancer cells to IFN-γ. In this regard, it is evident how profound the impactof epigenetic deregulation on tumor development may be, not only in termsof gene silencing of tumor-associated genes but also in terms of changes inthe biological behavior of a specific cell compartment. More recently, p21WAF1hypermethylation, taken as positive control of aberrant methylation in RMS cells,has been confirmed (Bott et al., 2005).

9.4.1.3 FGFR1. FGFR1 , the receptor of the growth factor FGF, has beenrecently reported to be overexpressed in RMS tumors. The importance of FGFR1signal transduction pathway in RMS tumorigenesis has been established in iden-tifying overexpression even of genes, such as AKT1, NOG , and its antagonistBMP4 , downstream to FGFR1 in primary RMS tumors with respect to normalskeletal muscles. Although evidence is lacking regarding genetic mutations in theFGFR1 coding sequence, analysis of the FGFR1 methylation pattern revealedthat a CpG island upstream to FGFR1 exon 1 was hypomethylated in the primaryRMS tumors, suggesting a novel mechanism for its transcriptional overexpression(Goldstein et al., 2007).

9.4.1.4 MGMT, CASP8, MyoD, HIC1, RB1, and CDX1. The MGMTmethyltransferase gene has been found to be hypermethylated only in a smallfraction of RMS samples (Yeager et al., 2003). Similar to neuroblastoma andmedulloblastoma, in which the CASP8 gene was frequently inactivated by acombination of methylation and allelic deletion (Zuzak et al., 2002) even in83% of RMSs, CASP8 was found to be aberrantly methylated (Rathi et al.,2003). As a matter of fact, using epigenetic profiling as a molecular marker to


distinguish RMS subclasses, it has been possible to evidence the preferentialhypermethylation of MyoD1 in ERMS (10 of 11; 91%), whereas it wasunmethylated in most of the ARMSs (13 of 15, 87%) (Siddiqui et al., 2003).In general, the MyoD1 gene was hypomethylated in RMSs compared to that innormal differentiated muscle (using methylation-sensitive restriction enzymes)(Chen et al., 1998). Moreover, in one case of ARMS in a 10-month-old boy,it was possible to screen the methylation status of 27 genes that are frequentlyhypermethylated in human cancers through methylation-specific polymerasechain reaction (PCR) analyses. Among these genes, promoter hypermethylationwas identified in only four TSGs: HIC1, HIN1, RB1 , and CDX1 , whereas therewas no detectable promoter hypermethylation for the myogenic marker MyoD1(Chang et al., 2005). Out of 22 primary cases of RMS, 13 (59%) showedhypermethylation of the HIC1 promoter by MSP (Rathi et al., 2003). Strikingly,100% (8/8) of ARMSs were hypermethylated, while hypermethylation was seenin a lower fraction of ERMSs, 33% (4/12), suggesting an important associationbetween HIC1 hypermethylation and tumor aggressiveness (Rathi et al., 2003).

9.4.1.5 HIN1. HIN1 (high in normal-1) encodes a putative cytokine that seemsto exert growth-inhibitory activities. While HIN1 methylation is rarely detected innonmalignant tissues (5%) (Shigematsu et al., 2005), hypermethylation-dependentloss of expression of HIN1 is a common event in many types of human malig-nancies. Although in some cases hypermethylation does not correlate with geneexpression downregulation, HIN1 promoter methylation has been completely cor-related with loss of expression. Analysis of the methylation status of the HIN1promoter revealed a high percentage (61%) of primary RMS tumors harboringHIN1 hypermethylation (Shigematsu et al., 2005).

9.4.1.6 Plakoglobin. The Plakoglobin gene encodes a connecting protein thatcooperates to link cadherin receptors to the actin cytoskeleton. In spite of induc-ing cell proliferation and tumor formation like β-catenin, plakoglobin promotestumor-suppressor activity. Interestingly, whereas this catenin has been found tobe expressed in ERMS cell lines and primary tumors, it is almost undetectablein ARMS, being restored by a combined treatment with AZA and TSA. At amore profound level, around the transcriptional start site of plakoglobin alleles,methylated CpG islands have been found only in ARMS cells and not in ERMScells (Gastaldi et al., 2006). This study presents plakoglobin as the first proteinwhose epigenetically regulated expression seems to be important in discriminat-ing between the two histological RMS subtypes. Epigenetic regulation of geneexpression has also been proposed to be utilized in anticancer therapy because itdoes not induce irreversible genetic changes. In a recent work, the TGFβ/Smadsignaling pathway, which is known to regulate RMS growth, was interrupted byusing an RNAi-mediated Smad4 silencing approach to suppress RMS growth andto strongly induce RMS apoptosis (Ye et al., 2006). This result implies that smallinterfering RNA may contribute to the epigenetic regulation of gene expression,being even applicable in rational intervention strategies in RMS therapy.


9.4.1.7 H19 and IGF2: The Imprinted Genes. H19 and IGF2 are known tobe affected by changes in DNA methylation in a number of childhood cancers,including Wilms tumors and ERMS (Dao et al., 1999). H19 encodes a growthsuppressor gene and IGF2 encodes a growth factor that plays a role in a vari-ety of malignancies. They are part of a cluster of imprinted genes on humanchromosome 11p15, which includes 57KIP2 , a cdk inhibitor that causes G1arrest; KvLQT1 , a voltage gated potassium channel; TSSC3 , a gene involvedin Fas-mediated apoptosis; and TSSC5 , a transmembrane protein-encoding gene(Feinberg, 1999). As the paternal copy of H19 is heavily methylated, its expres-sion results exclusively from the maternally inherited unmethylated allele. Con-versely, IGF2 is expressed only from the copy of chromosome 11 inherited fromthe father. The expression of these two genes is reciprocally regulated (Fig. 9.7).In fact, the chromosome region located between H19 and IGF2 genes representsan insulator region (or imprinting control region, ICR), which is unmethylated onmaternal chromosomes. Unmethylated ICR is bound by the CCCTC-binding fac-tor (CTCF), thus leading to the formation of a chromatin barrier that prevents theactivation of IGF2 by an enhancer located downstream of the H19 gene (Leunget al., 2001). On the contrary, the ICR on the paternal chromosome, being usuallymethylated, is not recognized by CTCF, thus leading to transcription of IGF2 . Inaddition, this normal imprinting chromatin status of both chromosomes permitsH19 to be expressed only by the maternal chromosome.

The fact that the 11p15.5 locus shows genetic alterations in human cancerand that this is frequently subjected to LOH in several pediatric cancers suggeststhe involvement of imprinted genes in tumor development. In tumors, methyla-tion of the normally unmethylated maternal H19 allele promotes the silencingof both copies of H19 , thus inducing IGF2 biallelic expression. One evidenceregarding the implication of H19 in RMS development was the observation thatH19 expression was significantly suppressed with respect to normal muscle tis-sue in 13 of 15 ERMSs and in 3 of 11 ARMSs (Casola et al., 1997). Removalof methylation from the maternal H19 gene by treatment of RMS cell lineswith a methyltransferase inhibitor causes activation of the silent paternal H19allele and a concomitant decrease in IGF2 expression (Chung et al., 1996). The

CTCFMat.

Pat.

Ins

Ins IGF2Ins-insulinIGF2-insulin like growth factorH19-designation of this non coding RNA transcript

IGF2 ICR H19

H19 Enhancers

Enhancers

ICR

FIGURE 9.7 Regulation of IGF2 and H19 imprinted genes. (From Jelinic and Shaw,2007.) (See insert for color representation of the figure.)


importance of methylation has been demonstrated in mice with decreased levelsof methylation, which showed expression of H19 from both alleles and a loss ofIGF2 expression (Krop et al., 2001). Preferential loss of the maternal 11p15.5alleles and loss of parental imprinting (LOI) are mechanisms that explain a gainof function of IGF2 in pediatric neoplasms, such as Wilms tumor, RMS, andhepatoblastoma. It has been reported that the number of functional IGF2 allelesis frequently increased in RMS, as a consequence of either LOI or gene dupli-cation. The maternal LOH commonly seen in tumors such as Wilms tumor andRMS eliminates the active maternal H19 gene and usually duplicates the activepaternal IGF2 gene (Cohen, 2005).

Besides the methylation pattern of H19 and IGF2 imprinted genes, lowerlevels of histone acetylation on their inactive alleles than on their active counter-parts have been reported (Schofield et al., 2001). Methylation and deacetylationare likely to be coupled mechanisms for gene silencing. It has been foundthat proteins that bind to methylated cytosines such as MeCP2, MBD1, andMBD2 can recruit HDACs (Hendrich and Bird, 2000). It has also been pro-posed that DNA methylation and histone deacetylation may act along the samepathway of inactivation. In fact, it has been shown that both the major DNMTenzyme DNMT1 (Vire et al., 2006) and the CTCF protein (Lynch et al., 2002),which binds the upstream insulator region between H19 and IGF2 and maycause histone deacetylation either directly or by interacting with HDACs, areinvolved.

In many cases, the reactivation of certain silenced TSGs caused by demethy-lating agents was augmented by the addition of HDACis such as TSA or thechemotherapeutic agent sodium butyrate (Weksberg et al., 2001). This raises thepossibility that a combinatorial therapeutic strategy may lead to efficient reactiva-tion of growth-inhibitory genes in certain cancers, including those that currentlyhave a poor response to chemotherapy such as RMS. In order to assess thepossibility of increasing the therapeutic efficacy in the treatment of RMS, theuse of a combined treatment, which both demethylates the gene and acetylatesits associated histones, has been proposed. Lynch and colleagues determinedif a combinatorial treatment of RMS cells with HDACis and inhibitors of DNAmethylation could cause a significant reactivation of a silenced H19 allele (Lynchet al., 2002). Nevertheless, they found that reacetylation of histones of the silencedH19 gene on its own was not sufficient to relieve repression and allow tran-scription, while the H19 gene could be reactivated readily using an inhibitor ofmethylation. Moreover, a combination of the two treatments carried out simulta-neously resulted in less reactivation than that seen for the methylation inhibitoron its own. Some evidences show that treatment of cells with a combinationof HDAC and methylation inhibitors could reactivate silent genes, which wereinstead unresponsive to the HDACis alone (Weksberg et al., 2001). Nevertheless,under most circumstances, the silent paternal H19 alleles cannot be reactivatedby treatment with HDACis on their own or in combination with demethylatingagents (Lynch et al., 2002). This indicates that methylation and not acetylationmay be the primary determinant of aberrant H19 silencing in RMS.

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9.4.1.8 TSSC5. TSSC5 (tumor-suppressing subchromosomal transferable frag-ment cDNA; also known as ORCTL2/IMPT1/BWR1A/SLC22A1L) encodes anefflux transporter-like protein with 10 transmembrane domains, whose regulationmay affect drug sensitivity, cellular metabolism, and growth. TSSC5 is locatedin a cluster region of human chromosome 11p15.5 and is subjected to paternalimprinting similar to the other genes of this locus. Aberrant methylation withinsome regions of the cluster that inhibits the correct function of ICR regions leadsto gene mysregulation or silencing. TSSC5 has been found to be downregulatedin certain tumors and has been involved in BWS, which is associated with suscep-tibility to Wilms tumor, RMS, and hepatoblastoma (Yamada and Gorbsky, 2006).

9.5 WILMS TUMOR

Wilms tumor (WT) is an embryonal solid neoplasia that occurs in childhoodwith an incidence of approximately 1 in 10,000 and is generally associated withovergrowth syndromes, such as BWS (Otte et al., 2004). It is a kidney cancer thatis believed to derive from progenitor cell populations of metanephric blastema(Rivera and Haber, 2005), which fails to terminally differentiate. Perilobularnephrogenic rests (PLNR) and intralobular nephrogenic rests (ILNR) are putativeprecursor lesions, showing persistent embryonal remnant tissues that differ inthe histologic features of the associated WT. This tumor is characterized byconstitutional genetic (somatic) alterations. Among these, LOH has been reportedin 32–38% of WTs (Fukuzawa et al., 2004; Yuan et al., 2005), especially at thelevel of chromosome 11p. On this chromosome, mutations of the WT gene atthe WT1 (11p13) and WT2 (11p15.5) loci have been reported. Other geneticalterations have been observed also at chromosome 16q, where LOH occursin 17% of WTs. The region (16q22) generally affected by this LOH containsthe gene encoding the CTCF protein that is implicated in the regulation of theimprinting status of the maternal H19 /IGF2 locus (Fukuzawa and Reeve, 2007).Nevertheless, other genes may be implicated in this instable chromosomal region.

As reported for rhabdomyoblastoma, LOI has been observed in WTs as anepigenetic abnormality associated with tumorigenesis. Genomic imprinting isa physiologic mechanism that allows a preferential expression of an allele inaccordance with its parental origin. Consequently, LOI involves aberrant activa-tion of the normally repressed parental inherited allele and is now considered tobe the most abundant and precocious alteration in cancer. In fact, monoallelicexpression is fundamental to ensure that the levels of the proteins encoded byimprinted genes do not exceed, avoiding developmental abnormalities (Jelinicand Shaw, 2007). Imprinting defects contributing to Wilms tumorigenesis havebeen recently observed at chromosome 11p13. In fact, WT1 expression wasshown to be affected by epigenetic disruption at regulatory regions in its locus.Specifically, one of the recent findings concerns the identification of a new classof epigenetically regulated noncoding RNAs, named WT1-AS , that originate atWT1 intron 1/exon 1 boundary. WT1-AS LOI causes their biallelic expression


and consequently the degradation of the WT1 target transcript (Hancock et al.,2007). The 11p15.5 chromosome comprises two independent imprinted domainsIGF2 /H19 and KIP2 /LIT1 , which frequently undergo maternal deletion or alter-ations associated with imprinting. LOI for the IGF2 gene was one of the firstepigenetic aberrations to be reported in WTs (Ogawa et al., 1993). Now it isconsidered to be an early event involved in the genesis of aberrant epigeneticmosaicism that predisposes to the onset of WT (Fukuzawa and Reeve, 2007).IGF2 LOI, which is due to hypermethylation of a DMR of H19 gene on mater-nal allele, has been detected in 36–50% of WTs (Fukuzawa et al., 2004; Yuanet al., 2005).

Human renal cancers differ markedly in children and adults. The mechanismsinvolved in the establishment of the epigenetic alteration generating IGF2 LOIin childhood and adult tumors are different. In WTs, IGF2 LOI prevalentlyderives from a gain of methylation instead, in adult tumors more imprinted lociof the 11p15 region have been observed to show a loss of methylation (Scelfoet al., 2002). Epigenetic alterations are not widespread from IGF2 toward theother imprinted flanking genes in WT (Bjornsson et al., 2007). This impliesthat imprinting is a modulated mechanism at the level of each specific clusterand that its disruption may represent a mark in cancer development. Epige-netic abnormalities have been correlated even with the putative precursor lesionsof WT. While ILNR is prevalently associated with mutations or deletions ofWT in chromosome 11p13, PLNR correlates with LOI at 11p15 encompass-ing the telomeric (IGF2 ) and centromeric (LIT1, antitranscript of the KvLQT1gene) imprinted domains (Fukuzawa and Reeve, 2007). Extending the analysisto other tumor-inducer genes, it has been highlighted that the involvement ofmultiple imprinted and stemness genes, such as PEG3, MEIS1 , and DLK1 , inthe expansion of the renal progenitor cell population, may cause and sustainWT (Dekel et al., 2006). Another gene correlated with Wilms tumorigenesis isHIN1 , which was found to be hypermethylated in 73% of tumors (Shigematsuet al., 2005). From a different point of view, data from methylation genomewideanalysis rather than studies restricted to CpG islands on promoter regions oftumor-associated single genes highlight the role of a global epigenetic distur-bance in tumors. An example is represented by satellite DNAs located at thecentromere-adjacent heterochromatin of chromosome 1 and 16, which were foundto be significantly hypomethylated in 35 WTs in comparison to normal post-natal somatic tissues (Qu et al., 1999). When this analysis was extended tocentromeric DNA throughout the genome’s chromosomes, 83% of the sampleswere hypomethylated. Interestingly, this loss of methylation was correlated withcytogenetically detectable chromosome rearrangements, suggesting that satel-lite DNA hypomethylation contributes to genome instability in WTs (Ehrlichet al., 2003).

The reciprocal contribution of genetic and epigenetic alterations in WT is stillnot known. Nevertheless, LOH of 11p15.5 and LOI of IGF2 represent the mostfrequent genetic (29%) and epigenetic (40%) alterations (Satoh et al., 2006).

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However, a comparative analysis showed that WT with LOI may be biologicallydifferent from those with LOH (Yuan et al., 2005).

9.5.1 Aberrant Methylation in Wilms Tumor

9.5.1.1 WT1, AWT1, WT1-AS. WT1 is a TSG located at the 11p13 chromosomethat encodes a zinc-finger transcription factor regulating early kidney develop-ment. Although WT1 was considered to be exerting a causative role, it is mutatedonly in about 10% of WT cases (Rivera et al., 2007). This apparent contradictionwas unraveled when different research groups studied WT1 gene silencing dueto epigenetic alteration. WT1 promoter hypermethylation was initially found tobe associated with LOH, a condition that fits with the “two-hits” model inacti-vation of a gene (Satoh et al., 2003). The coding region of this gene containsthe AWT1 gene, which encodes an alternative transcript of WT1 , and the WT1-AS gene, which encodes a family of antisense transcripts, previously found toregulate WT1 protein levels. Both of these are identified to be expressed fromthe paternal allele, with the silent maternal allele retaining methylation at thelevel of one WT1 antisense regulatory region (ARR). The ARR DMR is definedas a new transcriptional silencer element acting on both the AWT1 and WT1-AS promoters. When this silencer is methylated, AWT1 and WT1-AS genes aretranscriptionally repressed.

Specifically, WT1-AS and its spliceoforms originate within WT1 intron 1 andextend up to exon 1. Their promoter is adjacent to the ARR that is normally dif-ferentially methylated on the parental alleles. The physiological regulatory role ofWT1-AS depends upon their ability to interact with the complementary region ofthe WT1 transcript, forming RNA : RNA duplex, thus downregulating its expres-sion. It has been previously reported that WT1-AS expression is epigeneticallyregulated by the methylation of ARR (Malik et al., 2000; Dallosso et al., 2007).In fact, ARR has been demonstrated to be a methylation-dependent silencer onthe WT1-AS promoter (Hancock et al., 2007). Differential allelic methylation ofthe ARR correlates with the monoallelic expression of WT1-AS in normal kidney.Conversely, in WTs, there is LOI for these transcripts caused by ARR loss ofmethylation (Malik et al., 2000; Dallosso et al., 2007). With respect to the adultkidney, WT precursor lesions (nephrogenic rests) representative also of the fetalkidney, show increase in methylation levels, together with biallelic expressionof AWT1 and WT1-AS, suggesting that imprint erasure occurs during Wilmstumorigenesis (Hancock et al., 2007). These findings define human chromosome11p13 as a new imprinted locus, which is target of the epigenetic lesion in WTs.

9.5.1.2 WTX. Another gene somatically deleted in WT has been recently iden-tified on the X chromosome and has been named WTX . It was found to bemutated in approximately one-third of WTs. Tumors with mutations in WTXlack WT1 mutations; WTX is inactivated only in one of the two alleles, butwhat is more interesting is that the inactivated allele is always the one on the


active female X chromosome and that on the unique active male X chromosome(Rivera et al., 2007). This represents a link between genetic gene inactivationand epigenetic regulation of gene expression in a tumor context. Data collectedfrom functional studies provide evidence of the tumor-suppressor activity ofWTX. Recently, a possible mechanistic explanation for the tumor-suppressoractivity of WTX has been proposed. In fact, through tandem-affinity proteinpurification and mass spectrometry, it has been discovered that WTX forms acomplex with β-catenin, AXIN1, β-TrCP2 (β-transducin repeat-containing pro-tein 2), and APC (adenomatous polyposis coli), which are downstream effectorsof the tumorigenesis-associated WNT signal transduction pathway. It is evidentthat WTX recruits β-catenin, promoting its ubiquitination and degradation (Majoret al., 2007). Conversely, no association was detected between β-catenin and WT1expression (Satoh et al., 2003).

9.5.1.3 IGF2/ H19. As mentioned in Section 9.4, the expression of theimprinted genes IGF2 and H19 at chromosome 11p15.5 is controlled by DMRs.Among them, H19 DMR, known also as the imprinting control region (ICR),is located 5′ to the H19 promoter and is methylated on the paternal allele. ICRcontains some CTCF target sites (CTSs), and is bound by CTCF only when itis unmethylated. In normal conditions, it occurs on the maternal chromosome,allowing the redirection of the activating effect of a downstream enhancer onH19 rather than on IGF2 (Fig. 9.7). LOI at the maternally inherited allele, bymeans of hypermethylation of ICR, results in the aberrant biallelic expressionof IGF2 , and represents the most common epigenetic alteration in WT.Microdeletions within ICR are associated with a high incidence of WT, becauseit seems that spacing of the CTSs on the deleted allele is critical for the gain ofabnormal methylation and penetrance of the clinical phenotype (Sparago et al.,2007). As a consequence of the IGF2 LOI, H19 is repressed on the maternalchromosome. The inactivation of the H19 gene is seen in preneoplastic kidneytissue, indicating that it is an early event in tumorigenesis (Cui et al., 1997).In WT, H19 repression and ICR hypermethylation are associated with LOI(biallelic expression) of IGF2 (Astuti et al., 2005). Another DMR characterizedby imprinting defects, the DMR0, is located 5′ to the IGF2 promoter. To date, itseems that DMR0 may show methylation changes on both parental alleles ratherthan in cis, as different reports give evidence for both the cases (Sullivan et al.,1999; Monk et al., 2006; Murrell et al., 2008). A recent work shows that theloss of methylation occurs at the paternal allele (contrary to other reports), whilegain of methylation occurs at the IC1 region. Indeed, by using conventionalbisulphite sequences, all primary WTs (n = 10) with hypermethylation at IC1showed hypomethylation at DMR0, while those with normal IC1 methylationhad normal DMR0 methylation (Murrell et al., 2008). Hypomethylation ofDMR0 has been reported and is associated to LOI in WTs (Sullivan et al.,1999). No IC1 deletion was detected in 40 sporadic WTs (Murrell et al., 2008).

More recently, new constitutional 11p15 abnormalities, one microinsertionand one microdeletion at the level of H19 DMR, have been identified as a new

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class of imprinting center mutations in a large cohort of patients affected by WT(Todd et al., 2004). This study was performed on blood lymphocytes from 437individuals, and the identification of the constitutional defects associated withepimutations in WTs gives very important clinically relevant advantages in theestimation of pathological features with a simple blood test (Murrell et al., 2008).

9.5.1.4 GTL2/DLK1. As epigenetic alterations in the 11p15.5 imprinted genecluster are associated with disordered imprinting of IGF2 and H19 , a new pro-file of epigenetic abnormalities have been identified at the level of 14q32 locus.This locus encompasses two reciprocally imprinted genes, DLK1 and GTL2 . Asdescribed for H19 , GTL2 is maternally transcribed and shows paternal allelepromoter methylation; similar to IGF2, DLK1 is paternally expressed. DLK1encodes a transmembrane protein that is likely to be involved in differentiationprocesses, whereas GTL2 is an untranslated gene. The analysis of the imprintedstatus of DLK1 gene in a cohort of 30 WTs reveals a complete lack of LOI,differently from IGF2 at a structurally similar H19/IGF2 domain. The level ofDLK1 expression does not correlate with its imprinting status, but is associatedwith myogenic differentiation (Fukuzawa et al., 2005). Even if the epigenetic con-trol of the two IGF2 /H19 and DLK1 /GTL2 loci seems to be strikingly similar,no DLK1 LOI was reported in WT. This finding implies that there are differentmechanisms of imprinting operating at the IGF2 /H19 and DLK1 /GTL2 domainsthat bring to 11p15.5 LOI as a nonrandom epigenetic error. The GTL2 promoterDMR has been reported to be hypermethylated in only one of the 40 primaryWTs (2.5%) (Astuti et al., 2005).

9.5.1.5 CDKN1C, KVLQT1, and LIT1. Besides the two telomeric imprintedgene clusters, IGF2 and H19 , the chromosomal region 11p15.5 containsCDKN1C (also know as p57kip2), KVLQT1 , and LIT1 (also known asKvLQT1AS) in the centromeric domain (Fosmire et al., 2007). The transcript,called LIT1 (long QT intronic transcript 1), is an imprinted antisense transcriptidentified within the KvLQT1 locus that is expressed preferentially from thepaternal allele. On the silent maternal allele, it is normally methylated at thelevel of an intronic CpG island, and complete loss of maternal methylation intumors suggests that antisense regulation is involved in the development oftumorigenesis. Nevertheless, eight out of eight WTs exhibited normal imprintingof LIT1 and five out of five tumors displayed normal differential methylation atthe intronic CpG island (Mitsuya et al., 1999).

9.5.1.6 GLIPR1/RTVP1. The GLIPR1 /RTVP1 (glioma pathogenesis-related1/related to testis-specific, vespid, and pathogenesis proteins 1) gene waspreviously reported to be silenced by hypermethylation in prostate cancer (Renet al., 2004). On the contrary, in WTs, an uncharacterized paralog of thisgene was found to be hypomethylated in a very high percentage of primarysamples. Specifically, by using COBRA across CpG islands, the GLIPR1 /RTVP15′-region was hypomethylated in 21 of 24 (87.5%) WTs relative to normal


tissues. Moreover, GLIPR1 /RTVP1 loss of methylation correlated with highexpression of the transcript product (Chilukamarri et al., 2007).

9.5.1.7 PAX2, SIX2, and WT1. A recent work suggests a link between Poly-comb activation and epigenetic alterations in WTs. The role of PcG (Polycombgroup) proteins in regulating gene expression by DNA methylation is well known(Vire et al., 2006). During normal development, nephric-progenitor genes, suchas WT1, PAX2 , and SIX2 , normally undergo silencing in adulthood. Their reac-tivation correlates with embryonic kidney malignancy. Data collected from thispaper indicate that Polycomb activation through epigenetic modification of thenephric-progenitor genes is likely to be involved in WT oncogenesis. Specifically,in a small cohort of primary tumors (five cases), it was found that PAX2, SIX2 ,and WT1 were significantly hypomethylated, as well as in fetal kidneys. On thecontrary, these genes were hypermethylated in the adult kidney, in keeping withthe completion of the differentiation process (Dekel et al., 2006).

9.5.1.8 CRABP-II. The cellular RA binding protein II (CRABP-II) is a tran-scription factor that, upon interaction with RA, translocates into the nucleus andregulates the expression of RA-mediated signaling genes. It has been found tobe upregulated in human cancers and WTs and to be a direct target of NMYCin NB (Liang et al., 2008). The expression of CRABP-II was restricted to tumorsamples, along with that of NMYC, but was undetectable in normal adjacenttissues. Moreover, CRABP-II expression was higher in advanced than in early-stage tumors (Liang et al., 2008). By using MSP and the sodium bisulfite DNAsequencing technique, a CpG island in the CRABP-II promoter was unmethy-lated in 18 out of 22 primary WTs and was partially methylated in the remainingsamples. On the contrary, it was either methylated or partially methylated in 20normal tissues and was unmethylated in other 3 (Liang et al., 2008). CRABP-IIdemethylation observed in primary WTs could be a consequence of the globalhypomethylation observed in cancers compared to normal tissues.

9.5.1.9 HACE1. This gene was originally identified by cloning thet(6;15)(q21;q21) translocation found at the 6q21 locus in a WT case (Anglesio etal., 2004) in an attempt to identify a putative TSG. Indeed, other chromosomalrearrangements have previously been reported in this region. HACE1 encodesa novel E3 ubiquitin ligase that is frequently downregulated in human cancers.This genetic lesion correlated with the downregulation of the protein. In asecond, more recent work, the epigenetic regulation of HACE1 was investigatedby extending the analysis to 26 WTs. Loss of HACE1 expression correlatedwith hypermethylation of a CpG island (CpG-177), located upstream of thepromoter. Specifically, this island was found to be hypermethylated in 73%of tumors (19/26) versus 35% (9/26) of normal samples (Zhang et al., 2007).These findings indicate that HACE1 is epigenetically inactivated in WTs with ahigh incidence.

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9.5.1.10 MCJ, TNFRSF12, CASP8, MGMT, p16INK4a (CDKN2a), NORE1A,p14ARF, and DAPK. Many genes have been investigated to determine theirmethylation status in WTs but the implication of their epigenetic assessmentin this kind of malignancy has not yet been clarified. MCJ and TNFRSF12were methylated in 90% and 65% of the analyzed cases respectively (Ehrlichet al., 2002). CASP8 and MGMT were methylated in 43% and 30% of 40 WTsrespectively (Morris et al., 2003) In Morris’s analyses, the incidence of MGMTmethylation was higher in WT (30%) than in adult renal cell carcinoma (RCC).Nevertheless, Harada and coworkers did not detect MGMT methylation in acohort of 31 WTs (Harada et al., 2002). The p16INK4a (CDKN2a) promotermethylation has been reported in advanced-stage tumors (Steenman et al., 1994;Taniguchi et al., 1995; Arcellana-Panlilio et al., 2002; Morris et al., 2003). TheNORE1 gene located at 1q32.1 is a member of the RASSF1 gene family and existsin three isoforms (NORE1Aα, NORE1Aβ, and NORE1B ), which have separateCpG islands spanning their first exons. NORE1A methylation occurs in a subset ofprimary WTs with an intermediate frequency (15%) along with other genes suchas p14ARF (15%) and p16INK4a (10%) (Morris et al., 2003). DAPK , or death-associated protein kinase, mediates apoptosis by IFN-γ signaling. Studies on itspromoter methylation showed only a relative percentage of WTs with DAPKhypermethylation (11% of 40 cases) (Morris et al., 2003), even if a completeabsence of methylation was reported for this gene by Harada et al. (Haradaet al., 2002).

9.5.2 Some Unmethylated Genes: RARβ, CDH1, CDH13, and HIC1

The RARβ2 gene maps to 3p24 and encodes nuclear receptors for the retinoids thatexert antiproliferative effect. Retinoids play an essential role in kidney organo-genesis (Burrow, 2000). Even if both RARβ2 and RARβ4 isoforms are frequentlymethylated in lung, breast, and prostate cancers (Harada et al., 2002; Rathi et al.,2003), they have not shown de novo methylation of their promoters either inWT or in NB (Harada et al., 2002; Morris et al., 2003). Twenty-five percent ofWTs that show chromosome 16q allele loss have a poor prognosis. The deletedregion contains genes for E- and H-cadherins , which are known to enhancetumor progression and invasion. No or very low levels of promoter methylationwere detected for CDH13 (0%) and CDH1 (3%) in a study of 40 primary WTs(Morris et al., 2003). The TSG HIC1 is located at 17p13.3, a region of fre-quent allelic loss in many tumor types. Unlike other pediatric neoplasms, suchas RMSs, MBs, and retinoblastomas, a low percentage (3%, 1 of 31) of WTsshowed hypermethylation of the HIC1 promoter (Rathi et al., 2003).

9.6 RETINOBLASTOMA

Retinoblastoma is an intraocular malignant tumor that originates in developingcells of the retina. It represents the most common tumor of the eye in childhood.It has an incidence of 1 in 20,000 live births in all human races (Suckling et al.,


1982). Both the alleles of the RB1 gene are necessary to be mutated for thedisease to occur (Friend et al., 1986; Chim et al., 2003). RB1 is important incell cycle regulation in most cells and is inactivated or mutated in many humancancers. Retinoblastoma may occur in both hereditary and nonhereditary forms.About 40% of patients show the hereditary form characterized by autosomaldominant inheritance. Generally, they develop retinoblastoma in both eyes(bilateral multifocal retinoblastoma), and are predisposed to the occurrence ofsecondary neoplasms throughout the body (Zeschnigk et al., 2004). These includebone and soft tissue sarcomas, malignant melanoma, and neoplasms of the brainand meninges (Murrell et al., 2008). Otherwise, the sporadic disease accounts for60% of patients, which have retinoblastoma in only one eye (unilateral retinoblas-toma); this nonhereditary retinoblastoma consists of a unifocal retinal tumor andinvolves no increased risk of other cancers. In hereditary retinoblastoma patientsare heterozygous for an RB1 mutation, whereas in sporadic unilateral retinoblas-toma both RB1 mutations are somatic events that initiate tumor development andtherefore they cannot be inherited (Lohmann et al., 1997; Klutz et al., 1999). Inpatients affected by bilateral retinoblastoma, a second mutation in the wild-typeallele leads to biallelic inactivation of the RB1 gene, thereby initiating thedevelopment of the tumor focus. Diagnosis of retinoblastoma is possible withinfive years of age, even if it generally occurs earlier, approximately at the ageof 11–22 months. Although retinoblastomas can be successfully treated in morethan 95% of cases (de Sutter et al., 1987), patients still require constant lifelongfollow-up for early detection of recurrent tumors or detection of secondarytumors (Balmer et al., 2006). In retinoblastoma, the inactivation of both copiesof the RB1 gene is generally due to genetic aberrations, such as LOH at theRB1 locus at chromosome 13q14. Besides genetic aberrations, it is clear thatepigenetic alterations are also involved in the pathogenesis of retinoblastoma.Epigenetic events such as promoter hypermethylation have been described ascausative agents for RB1 functional inactivation (Sakai et al., 1991; Schutteet al., 1997; Zeschnigk et al., 1997; Choy et al., 2004). RB1 can also functionallyinteract with a large number of important proteins involved in heterochromatinbiology, such as HP1, Suv39h1, DNMT1, and factors of the Ssw1–snf1chromatin remodeling complex (Siddiqui et al., 2007). Therefore, a directconsequence of RB1 loss is chromatin modifications. In retinoblastoma, besidesRB1 , other TSGs and DNA repair genes have been shown to be frequentlyinactivated because of aberrant methylation at the 5′ promoter regions includingRASSF1A (59%) (Choy et al., 2004) and MGMT (35%) (Rathi et al., 2003).

9.6.1 Aberrant Methylation in Retinoblastoma

9.6.1.1 RB1. In 80–90% of both familial and sporadic retinoblastoma, RB1has been found to be inactivated (Bookstein and Lee, 1991). It has been observedthat in 10% of the retinoblastoma patients the cause of inactivation of theRB1 functions is the hypermethylation of the CpG-rich islands in the promoterregion of the RB1 gene, which is normally unmethylated in nontumor cells

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(De Falco and Giordano, 2006). Methylation of the RB1 gene is more commonin nonheritable than in heritable retinoblastoma (Corson and Gallie, 2007). Eventhough hypermethylation of the RB1 gene has been suggested by a few studies(Greger et al., 1994; Meier et al., 2007; Sampieri et al., 2006), it does not seemto be the major determinant for development of retinoblastoma tumors.

9.6.1.2 MGMT. MGMT exerts a DNA repair function that protects cells frommutagenic and cytotoxic O6-alkylguanine lesions caused by carcinogens, andits loss of function has been associated with tumor formation. As the absenceof MGMT has been observed to be rarely caused by deletion, rearrangement,or mutation (Dreijerink et al., 2001), it has been hypothesized that it may besubjected to an epigenetic control. In fact, previous studies have shown thathypermethylation of discrete regions of the MGMT CpG island were responsiblefor the absence of MGMT in human neoplasia and in glioma cell lines (Costelloet al., 1994; Vire et al., 2006). Accordingly, MGMT protein was frequently absentin human retinoblastoma tissue (Choy et al., 2004). In retinoblastoma, the occur-rence of epigenetic lesion in MGMT by hypermethylation of the promoter wasshown for the first time in 2002 (Choy et al., 2004). Through MSP analysis, aber-rant methylation of MGMT locus was observed in 8 out of 23 retinoblastomatumors (35%) with no expression of the MGMT protein. Moreover, these authorsfound a positive correlation between the MGMT hypermethylation level and thepoor tissue differentiation state of the retinoblastoma samples (Choy et al., 2004).Indeed, hypermethylation of the MGMT promoter was more frequently identifiedamong undifferentiated cases (60%) than in differentiated tumor samples (40%)(Choy et al., 2004), suggesting that increased methylation at the MGMT pro-moter may be associated with the younger patients. These results, together withthe observation that no MGMT promoter methylation was detected in normalretina tissue, suggest that silencing of MGMT by methylation of the promoterand reduced expression of MGMT may play an important role in the develop-ment and progression of retinoblastoma. In clinical applications, impaired MGMTexpression due to its hypermethylated promoter makes patients with retinoblas-toma sensitive to alkylation-based chemotherapy and represents a marker for theoccurrence of cancer and for prognostic therapeutic indications.

9.6.1.3 RASSF1A. The epigenetic silencing of the RASSF1A TSG, due topromoter hypermethylation, commonly occurred in (40–72%) lung cancer,(66.7%) nasopharyngeal carcinoma, (62%) primary mammary carcinoma, (59%)retinoblastoma, and (45%) adenocarcinoma of the uterine cervix (Choy et al.,2004). RASSF1A is located at 3p21.3 and encodes a 39 kDa predicted peptidewith a Ras-association domain. It is rarely mutated and LOH at 3p or 3p21.3 isnot common in retinoblastoma (Dreijerink et al., 2001). The epigenetic silencingof the RASSF1A gene, by promoter hypermethylation, was reported to occur in59% (10/17 cases) of retinoblastomas (Choy et al., 2004). In another study, ahigher percentage (89%, 17/19) of retinoblastoma primary tumors was found toharbor methylation at CpG islands of the RASSF1A gene (Cohen et al., 2008).


Choy et al. investigated the relationship between promoter hypermethylationof RASSF1A and MGMT in 40 bilateral and 28 unilateral retinoblastomacases. They detected promoter hypermethylation of RASSF1A in 56 of 68(82%) human retinoblastoma tumors regardless of age at presentation, sex,laterality, histology, family history, tumor staging, or optic nerve involvement.Importantly, RASSF1A was not hypermethylated in normal and nonmalignantretinal tissues adjacent to the tumor tissues in retinoblastoma specimens, clearlyindicating that promoter hypermethylation of RASSF1A occurs specificallyin cancer cells. Further, the RASSF1A gene was reactivated and was able toexpress RASSF1A transcript after treatment with 5-AzadCdR, which inhibitedDNA methylation. No significant alterations in cell cycle or apoptosis that wereassociated with the reactivation of RASSF1A were found (Choy et al., 2004).

9.6.1.4 CASP8, MLH1, HIC1, NEUROG1, SOCS1, IGF2, CACNA1G, andRUNX3. Methylation of the CASP8 gene promoter has been evidenced in10 of 17 primary tumors; the loss of function of this important proapoptoticgene is probably correlated to Fas-mediated apoptosis and retinoblastoma cellgrowth (Rathi et al., 2003). The methylation status of the DNA mismatch repairgene MLH1 was studied in 51 retinoblastoma tumors and 2 retinoblastomacell lines; data obtained demonstrated a high frequency of hypermethylation(67%, 34/51), providing evidence for the critical role played by MLH1 in thedevelopment of retinoblastoma. In addition, it seems that hypermethylation doesnot correlate with MSI (Choy et al., 2004). By MSP, 6 of 9 (67%) primary casesof retinoblastomas were found to be hypermethylated at the level of the HIC1promoter (Rathi et al., 2003). The Neurogenin 1 (NEUROG1 ) gene was foundto be hypermethylated in 52% of retinoblastomas (10/19) (Cohen et al., 2008).Conversely, SOCS1 (suppressor of cytokine signaling 1), IGF2 (insulin-likegrowth factor 2), RUNX3 (runt-related transcription factor 3), and CACNA1G(α-1 G T-type calcium channel gene) genes were not found to be methylated in19 cases of retinoblastomas (Cohen et al., 2008).

These first findings regarding the aberrant hypermethylation of some genes,such as RB1, MGMT, RASSF1A, MLH1 , and CASP8 , suggest the important roleof epigenetic mechanism in retinoblastoma progression, and indicated RASSF1Agene as being the most common target of epigenetic changes that is known inhuman retinoblastoma.

9.7 PRIMARY CENTRAL NERVOUS SYSTEM MALIGNANCIESIN CHILDHOOD

Although cancer is rare in children, primary malignancies of the CNS are themost frequent solid tumors and the second most frequent tumor pathology inchildhood. However, metastatic disease is relatively uncommon in children incontrast to CNS tumors in adults. Usually, 2- to 12-year-old children showtumors located prevalently in the posterior fossa (subtentorial region), whereasin infants and adolescents, equal frequencies are observed in the subtentorial and

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the supratentorial regions. Histologically, the two major types of pediatric brainmalignancies are either glial-cell-derived or neuroectodermal-derived tumors.The first are believed to evolve from normal glial cells, whereas the latter arefrom primitive neuronal-restricted progenitor cells. Glial-cell-derived tumors aretermed gliomas and include CNS malignancies composed mostly of malignantastrocytes (astrocytomas), ependymal cells (ependymomas), and oligodendroglialcells (oligodendrogliomas) that are less frequent in children than in adultpatients. Gliomas are graded according to the World Health Organization (WHO)criteria as grade I to IV, grade I being generally benign and the others showingprogressive malignancy. Ependymomas are stratified as grade I to grade IIItumor subtypes. Among the astrocytomas, patients with the grade IV tumor,named glioblastoma multiforme (GBM), have a median survival of less than oneyear. In children, most of the primary astrocytomas at diagnosis are of grade Iand it is believed that, except for a few ex novo cases, pediatric GBM evolvesfrom lower-grade tumors. The most aggressive CNS tumors in infancy derivefrom primitive neuroectoderma and are indicated medulloblastoma (MBs) whenthey arise in the cerebellum (infratentorial), and supratentorial primitive neuroec-todermal tumors (sPNET) if they occur in the brain. The clinical treatment ofpediatric CNS tumors highly affects the quality of life of young tumor survivorsbecause of toxic side effects during the critical phase of growth. Thus, despiteimproved therapies, many children who survive suffer from severe neuropsy-chological sequelae. Several clinical differences have been described betweenadult and childhood CNS malignancies, including tumor location and higherlethality in young patients. Compared to adult patients, the response to therapyof pediatric oncologic population is characterized by lower responsiveness incase of ependymoma and higher responsiveness in case of GBM.

Recently, a large body of evidence indicates that epigenetic gene regulationplays a major role in tumor pathogenesis. Promoter hypermethylation of geneswhose functions are known to be related to cancer progression and/or located atcritical chromosomal rearrangement regions were investigated by several groupsin the past years. Owing to the low frequency of CNS tumors in the pediatricpopulation, the literature on epigenetic aberrations in this group is relativelypoor. Most of the studies on epigenetic abnormalities have been conducted ontumors from adult patients and, often, the same genes have been shown laterto play a role in pediatric tumors. Conversely, several tumor suppressor genes(TSGs) have been evaluated for hypermethylation in adults, but have not beentested yet in children.

The two big families of glial-derived and neuroectodermal-derived tumors aredifferent in their origin, aggressiveness, and responsiveness to therapy. Thesefeatures have been related to epigenetic aberrations of some genes. Even if thesegenes may be different within the same tumor family, they can be helpful in theselection of a tailored therapy.

In this section, we report results from epigenetic studies on the glial-derivedtumors, such as astrocytoma, GBM, ependymoma, and oligodendroglioma, andon the neuroectodermal-derived group, such as MB and sPNET.


9.8 PEDIATRIC GLIAL-CELL-DERIVED TUMORS

9.8.1 Astrocytoma

Astrocytomas are believed to arise from precursors of normal astrocytes byacquisition of genomic aberrations, leading to malignant phenotype. While adultastrocytomas affect particularly cerebral hemispheres, the most frequent intracra-nial sites for pediatric astrocytomas are the cerebellum, the optic pathway, and thehypothalamic region. On the basis of WHO classification, they are graded fromgrade I to grade IV. The most common histological subtype in childhood is thelow-grade juvenile pilocytic astrocytoma (PA, grade I), considered to be benignand often found to be associated with neurofibromatosis type-1 (NF1) (Zhanget al., 2002). Diffuse astrocytomas in children include the low-grade fibrillary,gemistocytic and protoplasmic astrocytomas (grade II), the intermediate-gradeanaplastic astrocytomas (AA, grade III), and the most aggressive form GBM,grade IV. In children, AA and GBMs occur rarely as primary lesions, unlike inadults, and usually derive from tumors of a lower grade. Primary and secondaryglioblastomas are astrocytoma subtypes that are histopathologically indistinguish-able, but it is now accepted that they represent distinct disease entities developingin patients at different ages, involving different genetic pathways (Ohgaki etal., 2004), showing different expression profiles (Furuta et al., 2004; Tso et al.,2006), and often differing in their response to treatment (Ohgaki and Kleihues,2005). In an attempt to understand genetic etiology, adult astrocytomas have beenextensively analyzed for gene alterations in the past years.

The efforts to discover genetic alterations in pediatric malignant astrocytomashave been hampered by their relatively low frequency; indeed, they constituteonly 5–10% of childhood intracranial tumors, but they have a worse outcomecompared to other types of brain tumors. A number of findings indicate thatyounger age is a favorable prognostic factor, suggesting biological differencesin pediatric astrocytoma compared to its adult counterpart. Clinical knowledgeregarding diffuse pediatric astrocytomas indicates that even if they are oftenhighly invasive and able to locally metastasize, they are highly sensitive totreatments, contrary to adult tumors (Ganjavi and Malkin, 2002). There are manydistinctive traits between pediatric and adult astrocytomas. Molecular analysishas shown that childhood malignant astrocytomas rarely exhibit deletion of thetumor-suppressor PTEN or amplification of the epidermal growth factor receptor(EGFR), which are both gene abnormalities typical of adult aggressive gliomas(Watanabe et al., 1996; Bredel et al., 1999; Raffel et al., 1999). However, p53mutations have been detected in pediatric astrocytomas as well as in adultsecondary GBMs that progress from lower grade lesions (Watanabe et al., 1996;Wetzel et al., 2005). In low- or intermediate-grade adult astrocytomas (grade IIand the majority of grade III), a relatively normal-appearing genome or rarelynumerical chromosomal changes are detected. On the other hand, all adultGBMs and a subset of AAs present a complex pattern of genetic abnormalitiesincluding loss of chromosomes 7, 9p, 11p, 13q, 17p, 19q, and 22q. This suggeststhat the increasing grade of malignancy in adult patients may be due to a process

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involving cumulative genetic alterations. As a matter of fact, loss of the entirechromosome 10 and rearrangement/amplification of the epidermal growth factorreceptor (EGFR) gene have been often observed and considered to be causativeevents during the evolution of AA in GBMs. Conversely, in pediatric astro-cytomas, recurrent cytogenetic abnormalities are already present in low-gradetumor type, such as grade I PA (von Wronski et al., 1992; Bhattacharjee et al.,1997; Zhang et al., 2002). Interestingly, the most aggressive forms of pediatricastocytomas, AA and GBM, show a very low incidence of structural changes in 1,9, 17, and 22 chromosomes as seen in adult counterparts. From this comparativeanalysis it emerges that, although deriving from similar progenitor cells, adult andpediatric astrocytomas show important quantitative and qualitative differences.

In an effort to identify new therapeutic targets, several groups have focusedtheir attention on epigenetic events that are believed to cooperate with othermechanisms in TSG inactivation. In particular, gene promoter hypermethylationin adult astrocytomas is the most studied epigenetic change considered to beresponsible for TSG silencing. At present, low frequency of gene mutations hasbeen detected in childhood brain tumors, thus substantiating the hypothesis thatepigenetic aberrations instead may play a role in the development of pediatricastrocytomas. Owing to the low incidence of astrocytoma in childhood, onlyvery few molecular studies are currently available on gene promoter methyla-tion for this type of malignancy. In this section, evidence on genes generallyfound unmethylated or significantly hypermethylated in pediatric astrocytomas isreported. Sometimes, the methylation state does not correlate with gene expres-sion, as in the case of the TSG NF1 , whose low or absent expression significantlyassociates with PA. NF1 expression does not seem to be dependent on methyla-tion, as this gene was found to be unmethylated in a cohort of 30 PA, a cohortcontaining 12 II grade astrocytomas and 3 GBMs (Ebinger et al., 2004), asreported by others (Horan et al., 2000; Luijten et al., 2000; Fishbein et al.,2005). Other genes involved in tumor development and maintenance, such asglutathione-S-transferase P1 (GSTP1 ), DAPK1 , and p14ARF , were unmethy-lated or less methylated in pediatric PA even if their expression was low orabsent. Conversely, in MBs, they are consistently methylated (Gonzalez-Gomezet al., 2003a), confirming that tumors of different tissue origins may be char-acterized by diverse specific genetic and epigenetic backgrounds. Further, theTSC1 (9q34) gene was scarcely expressed in 50% of 50 pleomorphic xanthoas-trocytomas, a rare type of juvenile astrocytoma. Mutational analysis revealedneither mutations nor promoter methylation, suggesting that other mechanismsare responsible for its silencing in this type of tumor (Wong and Weber, 2007).Some cases of gene hypermethylation are reported below, together with a casein which hypomethylation instead of hypermethylation of a gene platelet-derivedgrowth factor B (PDGFB) affects cell responses (Bruna et al., 2007).

9.8.1.1 Aberrant Methylation in Astrocytoma

LATS1 and LATS2. The LATS (large tumor suppressor) gene encodes a ser-ine/threonine kinase (Justice et al., 1995; Xu et al., 1995). It was identified


in Drosophila as a TSG. Even in mammals, the two homologs LATS1 (largetumor suppressor, homolog 1) and LATS2 (large tumor suppressor, homolog 2)seem to play a role in tumorigenesis. In gene overexpression experiments, LATS1induced G2–M arrest and apoptosis in breast cancer cell lines in vitro and in vivo(Ren et al., 2004; Meng et al., 2006) and LATS2 caused G1–S arrest in NIH/v-ras-transformed cells in vitro and blocked tumorigenicity in vivo (Krop et al.,2001). Further, LATS1 knockout mice developed soft tissue sarcomas or ovariantumors (St John et al., 1999). These results suggested that these genes behaveas tumor suppressors in mammals. LATS1 promoter hypermethylation has beenreported in soft tissue sarcomas in parallel with mRNA decrease (Agirre et al.,2006). Hypermethylation of the promoter region of both genes was found tobe related to decreased mRNA expression in ALL and in breast cancer (Agirreet al., 2006; Uchida et al., 2006). Recently, in a cohort of 88 astrocytomas,the promoter hypermethylation frequencies of LATS1 and LATS2 were found tobe 56/88 (63.66%) and 63/88 (71.5%), respectively (Meng et al., 2006). In 10normal brain samples, where the two genes were well expressed, no methyla-tion was observed, indicating that this epigenetic change was specifically tumorrelated. In this study, samples were obtained from 23 astrocytomas of grade II,20 of grade III (AA), and 45 of grade IV (GBMs). Because the pediatric sam-ples represented a small portion of the entire cohort, their specific percentageof methylation was not evaluable. Nevertheless, this study gives informationon the expression of LATS1 and LATS2 in astrocytomas. The hypermethyla-tion frequency was found to increase in accordance with the severity (grade) ofthe tumor for both genes even if not always with significant difference amonggroups. This finding could indicate the more likely predisposition of these genesto be methylated in aggressive brain tumors. No correlation between the methyla-tion grade and age was found. LATS1 and LATS2 mRNA expression was lowerin all tumor specimens than in normal tissues, irrespective of their histologi-cal grade. This could be explained because the hypermethylation status did notinduce complete gene silencing in any tumor tissue even though significantlylower LATS1 and LATS2 mRNA expression was detected by real-time PCR inhypermethylated samples. Finally, in two astrocytoma cell lines, treatment with5-aza-deoxycytidine restored LATS1 and LATS2 expression, confirming that lowexpression is related to epigenetic modifications.

MGMT. The MGMT is an enzyme involved in repairing damaged DNA. Itremoves alkyl groups from the O6 position of guanine in DNA at the levelof the CpG sites. This function prevents G : C to A : T transition, favored by thebase modification, thus counteracting the action of DNA alkylating molecules.As a consequence of MGMT activity, the effects of chemotherapy with alkylatingmolecules, often used in the multimodal treatment of astrocytoma, are relieved.Thus, MGMT gene silencing by promoter hypermethylation seemed potentiallyrelated to increase in responsiveness to chemotherapy, being considered a positiveprognostic factor in adult astrocytomas (Knudsen et al., 1998; Hegi et al., 2004,2005). In spite of the low five-year survival rates ranging from 5% to 15% in chil-dren with GBM, alkylator-based chemotherapy associated with radiotherapy and


surgery strongly enhances survival time in most of the patients. When irradiationwas associated with alkylating agents, such as temozolomide, patients showedlonger survival than those treated with irradiation alone (Stupp et al., 2007). Theseclinical findings encouraged researchers to explore MGMT expression and methy-lation status in pediatric GBM. MGMT expression has been recently investigatedby immunohistochemistry in a large cohort of children with malignant gliomas(Wetzel et al., 2005). The authors observed that MGMT overexpression correlatedwith adverse outcome and that progression-free survival was significantly higherin patients with low-MGMT-expressing tumors. Recently, promoter hypermethy-lation of MGMT has been investigated in a cohort of 10 pediatric GBM patients,7 of whom had received temozolomide treatment (Donson et al., 2007). PartialMGMT promoter methylation was detected by MSP in 4/10 (40%) GBM tumors,all from patients treated with temozolomide. None of the samples showed com-plete methylation. Partial methylation was also observed in 4/5(80%) GBM celllines. A significant difference in survival time between children showing MGMTpromoter methylation and those with unmethylated MGMT was reported to be13.7 ± 3.7 versus 2.5 ± 1.7 months, respectively. In the subset of patients treatedwith temozolomide, the OS was significantly higher for four out of seven patientsdisplaying MGMT methylation (13.7 ± 3.7 vs 3.2 ± 1.9 months, respectively).Interestingly, a short-term cell culture established from a patient treated withtemozolomide showed MGMT methylation, whereas the original tumor samplewas unmethylated. This finding indicates that tumor cells in culture can changetheir epigenetic background and, therefore, they have to be carefully monitoredon the basis of primary tissue characteristics. Even though the number of pediatricpatients was less compared to that reported in studies on adults, the proportionof MGMT -methylated tumors was similar. This suggests that the better respon-siveness to alkylating chemotherapy in pediatric GBMs could be not relatedto MGMT methylation status. As reported by two independent studies, in PA(grade I) the frequencies of MGMT methylation ranged from 8% (1/13 sam-ples) to 25% (6/23), the increase in frequency being correlated to the age of thepatient (Gonzalez-Gomez et al., 2003a). Taken together, these data could implythat MGMT promoter hypermethylation may be a prognostic positive survivalfactor in pediatric GBMs. In contrast, the group of Matsuda showed that in diffuselow-grade astrocytomas, the low expression of MGMT was related to malignanttransformation (Nakasu et al., 2007). Thus, the role of the epigenetic downreg-ulation of MGMT in astrocytomas could be related to tumor grade and remainsto be elucidated. As a matter of fact, a link between MGMT hypermethylationand p53 mutations has been established in pediatric astrocytomagenesis. Indeed,p53 function is necessary for DNA repair and is often mutated in tumors. Thedecreased activity of MGMT in neutralizing mutagen-induced G : C to A : Ttransition, due to hypermethylation, is believed to be responsible for the highfrequency of p53 mutations in PA, low-grade adult astrocytomas, and secondaryrather than primary adult GBMs (Nakamura et al., 2001b; Gonzalez-Gomez et al.,2003a; Ohgaki et al., 2004; Yoshino et al., 2007). In pediatric GBMs, p53 muta-tion is a frequent event compared to that in adults and has been correlated with


low survival time (Watanabe et al., 1996; Wetzel et al., 2005). It is thus possiblethat p53 mutations, together with their loss of functions, are due to DNA adductsin MGMT target regions in tumors with silenced or lowly expressed MGMT.

p14/ARF/p53 Pathway. The INK4a/ARF locus, located at the 9p21 chromosome,encodes two proteins with tumor-suppressor activity, p14ARF and p16INK4a ,in different reading frames. These two proteins are different in amino acidsequence and act on distinctive regulatory pathways. p14ARF induces degra-dation of MDM2, thus hampering MDM2 binding to p53, which consequentlyaccumulates into the cells triggering G1 and/or G2–M cell-cycle arrest (Lindseyet al., 2004). Loss of p14ARF function has been observed in several types of can-cers. Recently, its promoter hypermethylation has been shown in astrocytomas.Nevertheless, the association of p14ARF methylation with the histological sub-types in astrocytomas of adults, and thus its correlation with outcome, remainscontroversial. p14ARF hypermethylation has been reported to be 15–50% inhigh-grade astrocytomas (Gonzalez-Gomez et al., 2003a) and 20% in low-gradediffuse astrocytomas, moreover being correlated with low, progression-free sur-vival (Watanabe et al., 2003). Further, high frequencies of p14ARF methylationwere shown in diffuse adult astrocytomas of grade II (33%) and in secondaryadult GBMs (31%), while it was lower in primary GBMs (6%) (Nakamura etal., 2001a). The methylation status correlated with low expression of p14ARFprotein detected through immunohistochemistry in all specimens. In children, acohort of 13 PA has been examined, and no methylation of the p14ARF promoterwas observed in any sample (Gonzalez-Gomez et al., 2003a). The role of the twoTSGs, p53 and p73, on the pathogenesis of PA is still unclear. One report showedp53 mutations in 7/20 (35%) PA pediatric patients (Hayes et al., 1999), whereasseveral other studies did not show any mutation of these genes in PA and/or inlow-grade juvenile astrocytomas. It is believed that p53 mutation represents anearly event in the development of adult astrocytomas and may be an indicator ofshort, progression-free survival when associated to p14ARF methylation (Watan-abe et al., 2003; Kreiger et al., 2005). A few data are available on the epigeneticdisregulation of p53 and p73 in astrocytomas. Low frequency of methylation inthe p53 promoter was detected in 1/13 (8%) PAs, whereas evaluation of the p73promoter evidenced methylation in 3/13 (23%) (Hayes et al., 1999).

p16/INK4a/RB1 Pathway. Cell proliferation is strictly controlled by a plethoraof molecules, each checking different phases of cell cycle progression. Amongthese, p16/INK4a plays a key role in the maintenance of cell growth control byinhibiting phosphorylation of RB1 through CDK4/cyclin D1 binding, thus pre-venting the G1–S transition in response to stress or oncogenic events (De Leonet al., 2008). p16/INK4a is expressed at low levels in normal adult tissues andincreases in an age-dependent manner, thus representing a molecular marker ofaging (Ohgaki and Kleihues, 2007). It has been shown that mice expressing lowlevels of p16/INK4a are prone to develop tumors (Sharpless et al., 2001; Lindseyet al., 2004). p16/INK4a functions are lost by mutation or promoter hypermethy-lation in several cancers (Lindsey et al., 2004; Collado et al., 2007). A study on


a cohort of 13 pediatric PA evaluating the methylation status of 10 tumor-relatedgenes by MSP reported a high methylation frequency of p16/INK4a (46%,6/13) compared to non-neoplastic cortex and white matter. Two cases of thempresented also methylation of RB1 , that on the whole resulted methylated in anadditional sample with unmethylated p16/INK4a samples (Gonzalez-Gomez etal., 2003a). RB1 methylation was also shown in GBMs and AA with frequenciesof 21% and 14% respectively (Gonzalez-Gomez et al., 2003a). The study onPA in children suggests that the p16/INK4a/RB1 pathway is predominantlyderegulated in these tumors. The methylation index (MI; number of genesmethylated/number of genes tested for each sample) was 0.18 in PA tumor series,while only three patients displayed no methylation for any gene tested, indicatingthat methylation could be a common event in these tumors. The prominent levelof p16/INK4a methylation in pediatric PA is in agreement with increased fre-quency of methylation in low-grade compared to high-grade adult astrocytomas,as reported by the same group in a second work (Gonzalez-Gomez et al., 2003b).No data are available on pediatric astrocytomas of grade III or IV for both genes.

THBS1 and TIMP3. Thrombospondin 1 (THBS 1) and tissue inhibitor of metal-loproteinases 3 (TIMP3 ) genes inhibit metastasis, invasion, and angiogenesis,acting on different pathways. THBS has been found to be methylated in severaltypes of cancers, suggesting that its silencing or downregulation may be a tumor-related event (Gonzalez-Gomez et al., 2003a; Ren et al., 2004). Similarly, TIMP3has been found to be methylated in different types of cancers such as colon carci-nomas, NBs, and MBs (Muhlisch et al., 2006; Berkhout et al., 2007; Michalowskiet al., 2008). The study of Gomez-Gonzalez and coworkers showed significantmethylation of these two genes in PA. Specifically, THBS showed 5/13 (38%)and TIMP3 4/13 (31%) methylation frequencies. In 4 of the 13 samples, bothwere methylated, in agreement with other studies on adult and pediatric gliomas(Hayes et al., 1999; Gonzalez-Gomez et al., 2003a).

CXCL12 (SDF1). Chemokines are chemotactic cytokines regulating several phys-iological and pathological processes such as immune response, mobilization ofhematopoietic stem cells, and tumor invasion (Balkwill, 2004). They are solublefactors that bind their receptors on target cells, thus stimulating transcription ofgenes involved in migration, invasion, interaction with extracellular matrix, andsurvival. CXCL12 (stromal derived factor-1, SDF1) is a cytokine produced phys-iologically by bone marrow and brain cells and pathologically by tumor cells.Its receptor, CXCR4, is present on a wide variety of cells, including white bloodand endothelial cells. High expression of CXCR4 is found in breast, colon, andmelanoma cancer cells (Agathanggelou et al., 2003; Ottaiano et al., 2005; Scalaet al., 2006). The role of this receptor on cells has been related to motility, as cellsexpressing CXCR4 are easily mobilized toward CXCL12-expressing tissues, aphenomenon that might explain the capability of tumor cells to metastasize to spe-cific target tissues. Deregulated CXCL12-CXCR4 signaling seems to be involvedin several types of malignancies such as breast, hepatocellular and basal cell car-cinomas (Schimanski et al., 2006; Maroni et al., 2007; Nunbhakdi-Craig et al.,


2007). In astrocytoma, tumor progression has been related to CXCL12-CXCR4cell-guided invasion (Bajetto et al., 2006), and in vitro inhibition of this pathwayarrested proliferation of glioma cell lines (Sehgal et al., 1998). To date, the sig-nificance of low expression of CXCL12 is not clear, but it could be supposed thatexpression of CXCL12 in primary tumors may sustain growth and survival butdiscourages invasion and metastasis, maintaining cancer cells in site. In a studyon 76 astrocytomas by mean of MSP (20 astrocytomas of grade II, 26 AA ofgrade III, and 30 GBM) in patients from 10 to 81 years of age, CXCL12 methy-lation was shown in 26/76 (34.2%) astrocytomas: in particular, in 11/20 (55%)low-grade astrocytomas, 9/26 (34.6%) AA, and 6/30 (20%) GBMs (Chang et al.,2005). No significant differences were found with regard to sex and age but aninverse correlation was found between CXCL12 methylation and tumor grade. Inmethylated astrocytomas (WHO II and III), lower expression of CXCL12 mRNAwas observed with respect to unmethylated cases, while in GBM no statisticaldifference in expression of CXCL12 was shown in methylated as compared tounmethylated cases (Chang et al., 2005). CXCL12 expression was higher in astro-cytomas than in normal brain, and increased in GBM, suggesting an importantrole in the progression of tumor state in agreement with reports indicating anoverexpression of these chemokines in malignant glioma (Salmaggi et al., 2004,2005).

CST6. Cystatin 6 (CST6) belongs to a family of proteins that are endogenousinhibitors of cathepsins. Cathepsins are lysosomal cystein proteases involvedin sustaining multiple steps of tumor progression such as escape from immunesurveillance (Watts et al., 2003) and angiogenesis (Yanamandra et al., 2004).Numerous studies have demonstrated a correlation between upregulation ofcathepsin B, D, and L and invasiveness and tumor grade of gliomas (Mohanamet al., 2001; Sivaparvathi et al., 1995, 1996). CST6 has been shown to befrequently downregulated in breast cancers, and its ectopic reexpression invitro reduces proliferation, migration, invasion, and adhesion (Shridhar et al.,2004). Epigenetic silencing due to hypermethylation of CTS6 promoter wasobserved in many types of tumors such as preinvasive breast cancer lesionsand ductal in situ carcinoma (Virmani et al., 2000), breast carcinoma metastaticlesions and cell lines (Rastetter et al., 2007; Rivenbark et al., 2007), cervicalcancer tissues and cell lines (Veena et al., 2008), and non-small cell lungcancer (Zhong et al., 2007). In 18 of 21 (75%) CTS6 low-expressing adultand pediatric glioma tumors, the gene was methylated along with that in 8 of12 (66%) pediatric-patient-derived tumor cell lines. Moreover, PAs showed alower frequency of CTS6 methylation (2/5 hemimethylated, 40%) than gradeII and III astrocytomas (4/5, 80%). Strikingly, ependymoma-patient-derivedshort-term cell cultures showed 100% (3/3) CTS6 hypermethylation. Treatmentwith 5-azadC restored CTS6 expression in grade II and III astrocytomas andependymoma primary cell lines, whereas downregulation of CTS6 was notaffected in PA. This finding implicates that the hemimethylation in PA was notthe mechanism responsible for CTS6 low expression, but that other mechanismsmay be involved (Qiu et al., 2008).


PDGFB. PDGFB is a potent mitogen and angiogenic factor (Fredriksson et al.,2004) that is implicated in gliomagenesis (Dai et al., 2001; Guo et al., 2003; Ost-man, 2004). PDGFB has been found to be methylated in about 50% of humangliomas with low expression of the gene (Bruna et al., 2007). Bruna and cowork-ers showed that TGFβ treatment promotes tumor cell proliferation by inducingPDGFB transcription (Bruna et al., 2007). Despite the antiproliferative role ofTGFβ in normal epithelial cells and astrocytes, in some malignant gliomas TGFβ

loses its tumor-suppressor activity, exerting oncogenic function (Seoane et al.,2004). As a matter of fact, it induces proliferation, invasion, and angiogenesis(Derynck et al., 2001; Siegel and Massague, 2003). To unveil what discrimi-nates the dual role of TGFβ as a tumor-suppressor or tumor-promoter factor,Bruna and colleagues identified PDGFB among the genes that were upregu-lated in glioma cells under TGFβ treatment. As low levels of PDGFB expressionappeared to correlate with the incapability of TGFβ to induce proliferation, themethylation status of PDGFB promoter was investigated by using MSP andbisulphite sequencing in 8 patient-derived glioma cells, human fetal astrocytes,normal human progenitor, and in 21 glioma tumor samples. Promoter methy-lation was found in 3 of 6 patient-derived glioma cell lines in which TGFβ

did not induce proliferation, and in 10 of 21 astrocytoma tumor samples withPDGFB low expression, in contrast to normal controls that were not methylated(Bruna et al., 2007). These results may suggest that the methylation status ofa gene may be effective in dictating tumor cell response to cytokine-inducedpathways.

9.8.2 Ependymoma

Ependymoma is a glioma that develops both in children and adults, thoughtto be derived from ependymal cells that line cerebral ventricles and the centralcanal of the spinal cord. Most of the adult tumors occur in the spinal cord,whereas pediatric tumors are more often intracranial. This represents the secondmost common malignant brain tumor in children and adolescents, and accountsfor 3–9% of all intracranial (supra- and infratentorial) and about 60% of spinaltumors. Children with ependymoma show a worse clinical outcome comparedto adults, with a more favorable prognosis for spinal tumors, which is relativelyrare in children and young adults (Lusser et al., 1999). Ependymoma is classifiedby WHO as myxopapillary ependymoma (grade I), subependymoma (grade I),ependymoma (grade II), and anaplastic ependymoma (grade III) on the basis ofhistopathologic characteristics. A variant of primitive neuroectodermal tumors(PNETs) is represented by ependymoblastoma, which is considered to be ahighly aggressive subtype. The prognosis is poorer in patients less than threeyears of age at diagnosis and/or with anaplastic tumors. Ependymoma is poorlyunderstood at the molecular level. Chromosomes 1q, 6q, 7, 9p, 10, 11, 13q,15q, 16q, 17p, 20p, and 22q have been described as presenting multiple regionsof gain and/or loss at different percentages. Intracranial and spinal tumors showcommon chromosomal abnormalities and also genetic differences, as mutations


of the neurofibromatosis type 2 (NF2 ) gene mainly present in spinal tumors,which may be responsible for different clinical developments (Hirose et al., 2001;Zhang et al., 2002, 2004; Fosmire et al., 2007). Nevertheless, the general lowlevel of gene mutations detected in childhood brain tumors suggests that othermechanisms such as epigenetic dysregulation can play a role in the pathogenesisof this disease. Thus, molecular researchers have started investigating promotermethylation of candidate genes known to be silenced by methylation in othertypes of cancers, to be tumor suppressors, and to be located at LOH-sensitivechromosomal regions in ependymoma. However, to date, few studies areavailable on epigenetic abnormalities found in ependymoma maybe for therarity of the tumor and the unavailability of ependymoma cell lines as a modelfor in vitro studies. Further, results from these studies are sometimes conflictingmaybe due to differences in the methods of detection and quantification, tothe small size of patient’s cohorts, and to the inaccessibility of normal tissuecounterparts for comparative studies (i.e., nonneoplastic ependymal cells). Also,the heterogeneity of cells within a tumor mass can determine controversialfindings as reported for infiltrating leukocytes as a source of methylation(Lombaerts et al., 2004). Apart from genes showing significant rates of aberrantmethylation at least in one study, candidate genes related to cell invasion such asDAPK and CDH1 , or to cell cycle control such as p15/INK4b, p14ARF, RARβ,RB1, FHIT , p73, and p53 were unmethylated or hypermethylated with lowfrequency (<10%) in subsets of ependymoma (Gonzalez-Gomez et al., 2003a;Rousseau et al., 2003; Waha et al., 2004; Gaspar et al., 2006; Michalowskiet al., 2008). Consequently, epigenetic alterations in these genes appear moreto be casual events than determinants of tumor phenotype. On the other hand,other genes have been found aberrantly methylated in ependymoma with highrecurrence, possibly representing tumor-associated features. Some of them arediscussed in the following paragraphs.

9.8.2.1 Aberrant Methylation in Ependymoma.

HIC1. A high incidence of LOH at chromosome 17p13.3 has been reportedto be common in pediatric astrocytomas and MBs (Steichen-Gersdorf et al.,1997; Wales et al., 1995). In this region, the gene coding for the transcriptionalrepressor HIC1 (hypermethylated in cancer 1) is located, whose hypermethy-lation associated with transcriptional silencing has been previously reported inadult astrocytomas, MBs, and other carcinomas (Wales et al., 1995; Kanai et al.,1999; Waha et al., 2003). Two studies indicate HIC1 as being highly methy-lated in ependymomas. In one study using MSP, HIC1 was methylated in 85%of 27 pediatric ependymomas and in 68% of 28 adult cases, in at least one ofthe two regions analyzed (promoter or central exon 2), highlighting age-relatedmolecular differences (Waha et al., 2004). All recurrent tumors showed HIC1hypermethylation, while in normal brain HIC1 methylation was reported onlyin <10% of the cases. Low or absent expression of the gene was observed bycompetitive RT-PCR in 80% of hypermethylated ependymomas, being expressedin the remaining 20%. A regional heterogeneity in methylation spanning the


promoter regions may be responsible for the different levels of HIC1 expression.Interestingly, HIC1 methylation was detected in a higher percentage (94%) oftumors with intracranial localization compared to that (65%) in tumors with spinallocalization, although they showed the same methylation pattern. This evidenceindicates that intracranial and spinal ependymomas could be genetically distincttumors as already suggested (Fosmire et al., 2007). In another study, Modenaand coworkers examined a cohort of 18 primary intracranial ependymomas usingMSP and found HIC1 promoter hypermethylation in 83% of the cases (Modenaet al., 2006). Both studies suggest that the aberrant methylation of HIC1 , beingwidespread in ependymomas, may contribute to the pathogenesis of this tumor.

RASSF1A. Among genes involved in cell cycle regulation, the TSG RASSF1A,aberrantly methylated in a variety of cancers of pediatric and adult origin, hasbeen found hypermethylated on its promoter in a significant fraction of ependy-momas by two different research groups (Hamilton et al., 2005; Michalowskiet al., 2008). One study using MSP and COBRA revealed a very high fre-quency of RASSF1A promoter methylation in 17 of 20 (85%) tumors belongingto a mixed cohort of 11 pediatric and 9 adult ependymomas (Hamilton et al.,2005). RASSF1A methylation seemed to be relevant because it was present inall histopathological and clinical tumor subtypes but not in nonneoplastic tis-sues. By bisulphite sequencing, the authors confirmed methylation of 11 of 14CpG residues analyzed in tumors and one methylated residue in only 1 of 6 nor-mal brain samples. These results indicate that RASSF1A hypermethylation is atumor-specific event. A frequency of 56.5% of RASSF1A hypermethylation wasassessed through MSP by Michalowski and coworkers in 27 intracranial pedi-atric ependymomas (Michalowski et al., 2008). Also in this study, normal cortexdid not present any methylation. Interestingly, in choroid plexus papillomas, abenign intracranial neoplasm of neuroepithelial origin lining the papillary projec-tion of the ventricular ependyma, the frequency of RASSF1A hypermethylationwas 67% (4 of 6 cases), although no statistically significant differences werereached between the two groups of tumors. But the cohort of benign tumors istoo small to be indicative.

CASP8. Michalowski and colleagues reported 30% hypermethylation of CASP8(caspase 8, cysteine-aspartic acid protease 8), a gene inducing apoptosis in PNETsof the central and peripheral nervous system (Michalowski et al., 2008). Only20% of hypermethylation was observed in the myxopapillary subtype of ependy-momas by Hamilton et al., while Alonso and coworkers detected <10% frequency(1/14 adult ependymomas in a cohort of 27 patients) (Gonzalez-Gomez et al.,2003a; Hamilton et al., 2005). Both studies utilized MSP and differed mainly inthe type of tissue used—frozen versus paraffin embedded—which can accountfor the difference in results.

p16/INK4a. The cell cycle regulator p16/INK4a has been subjected to intenseinvestigation for its key role in cancer development. Its point mutations, hyperme-thylation, and deletion are noticeable events in many types of cancers. p16/INK4a


together with p15/INK4b and p14ARF are located on chromosome 9, and all acton the G1–S and G2–M cell cycle transitions via RB1 and p53 pathway reg-ulation. The association of p16/INK4a hypermethylation to tumor grade is stillambiguous. Indeed, results on p16/INK4a epigenetic changes as determinants ofependymoma pathogenesis are still controversial. A cohort of 123 ependymomasfrom grade I to grade III was analyzed by Rousseau and colleagues (Rousseauet al., 2003). They reported 21% frequency of methylation, using MSP, irre-spective of the grade of tumor, location (intra- or extracranial), and age. Alonsoand coworkers showed more than 20% of p16/INK4a methylation in low-gradetumors from 10 adults and 12 pediatric patients, while p16/INK4a was not hyper-methylated in 5 high-grade anaplastic tumors (Gonzalez-Gomez et al., 2003a).No methylated templates were found in two nonneoplastic brain tissues. The lowfrequency of p16/INK4a hypermethylation in low-grade (grade II) tumors (11%)was detected in two different studies (3/27 and 2/18, respectively). No methy-lation was seen by two researcher groups (Vogelstein et al., 1985; Bortolottoet al., 2001). A recent study on a small cohort of patients reports detection ofp16/INK4a hypermethylation, confirmed by bisulphite sequencing and COBRAanalysis, only in 1/4 anaplastic tumor specimens (25%) and no methylation inlow-grade tumors (Muhlisch et al., 2006). Taken together, these data seem tosuggest that silencing of p16/INK4a by promoter methylation is an additionalmodification that could only cooperate in supporting ependymoma pathogenesis.

THBS1 and TIMP3. Studies on the two inhibitors of angiogenesis and invasion,THBS1 and TIMP3, reported variable frequency of methylation of their genes.The group of Rey, using MSP, has found both THBS1 and TIMP3 consis-tently hypermethylated (>20%) in 13 low-grade and 1 anaplastic ependymomas(Gonzalez-Gomez et al., 2003a). In particular, the hypermethylation frequency ofTIMP3 was 30% (4/13) and that of THBS1 was 38% (5/13) in grade II pediatricependymomas. Both genes showed methylation in the anaplastic form. Con-versely, two studies have found no methylation of TIMP3 by MSP in two cohortsof 11 and 27 patients with low-grade and anaplastic ependymomas (Vogelsteinet al., 1985; Michalowski et al., 2008). Finally, Muhlish and colleagues reported12.5% methylation by MSP and 0% by COBRA analysis in pediatric anaplasticependymomas (Muhlisch et al., 2006).

MCJ (DNAJD1). Members of the DNAJ proteins are involved in biologicalprocesses such as regulation of protein kinases, protein folding, and proteintranslocation (Maze et al., 1997). Their loss determines unresponsiveness to invitro chemotherapeutic agents and is related to their response to chemother-apy and to the OS in patients with ovarian cancers (Strathdee et al., 2005).Recently, Lindsey and coworkers reported results on methylation of the MCJgene in MB, stPNETs, and ependymomas, which together represent the mostcommon brain malignancies in childhood. They showed extensive hypermethy-lation in 2/20 (10%) primary ependymomas by COBRA analysis and bisulphitesequencing (Lindsey et al., 2004). Every CpG site was methylated in one sample


and unmethylated sites interspersed with methylated ones were shown by theother sample. No association was possible with the histopathological subtype orlocation. In contrast to the normal ovarian epithelial tissue exhibiting hyperme-thylation of MCJ, the four cerebellar and seven cerebral normal brain tissuesexamined showed no methylation, suggesting the role of a tumor-specific eventfor MCJ methylation in ependymomas.

MGMT. Low levels of O6-methylguanine methyltransferase (MGMT) proteinhas been related to a better prognosis in pediatric CNS tumors as its deficiencyincreases the susceptibility to chemotherapeutic agents such as nitrosoureas ortemozolomide (Pollack et al., 2003). Alonso and coworkers showed an aberranthigh rate of MGMT methylation in 2 of 10 (20%) low-grade adult and 3 of 12(25%) pediatric tumors, and in 2 of 4 (50%) adult and 0/1 (0%) pediatric anaplas-tic ependymomas (Gonzalez-Gomez et al., 2003a). A methylation frequency of5% (1/20) in a cohort of 11 pediatric and 9 adult ependymomas was reportedin another study (Vogelstein et al., 1985). In accordance with this last work,only 1 sample in a cohort of 27 ependymomas of grade II showed methylation(Michalowski et al., 2008). Finally, in 24 intracranial ependymomas, no methy-lation of MGMT gene was detected (Modena et al., 2006). All these studies wereconducted by MSP. These findings indicate that MGMT methylation could notrepresent a molecular event associated with pediatric ependymoma.

9.8.3 Oligodendroglioma

Oligodendrogliomas (OD) arise from white matter of cerebral hemispheres mainlyof the frontal lobes, rarely metastasize outside of CNS, and constitute 5–20%of gliomas. Pediatric ODs are rare tumors and represent only 1–3% of pediatricCNS neoplasms (Dohrmann et al., 1978; Razack et al., 1998). ODs predomi-nantly develop in adulthood with a global 5 years survival rate of 51%, whichdecreases with age. Patients with low-grade OD survive for 10–15 years witha 5-year survival rate of 75% (Shaw et al., 1992; Leighton et al., 1997). Low-grade OD (WHO grade I and II) is a diffusely infiltrating tumor composed ofwell-differentiated oligodendroglial-like cells. Anaplastic OD (AOD, WHO III)is believed to gradually evolve from low-grade OD, showing a less favorableprognosis with respect to the tumor of origin and, to date, a clear distinctionbetween low and high grades is missing.

Another type of OD consists in oligoastrocytoma (OA), which has an inter-mediate prognosis. Even if pediatric ODs appear histologically similar to adultones, they are more aggressive. ODs have a better prognosis than astrocytomaseven if their outcome is often fatal. Owing to the rarity of ODs in children, fewstudies have described the histopathologic and molecular features of this tumor.

The genetic aberrations typical of a subset of adult ODs are 1p and 19qchromosome losses, and they appear to positively correlate with the response tochemotherapy (van den Bent et al., 2008). Raghavan and coworkers found thatloss of 1p and 19q in ODs occurs rarely in childhood, particularly in the first


decade. Indeed, none of 15 patients younger than nine years showed these losses,whereas patients older than nine years showed 1p (45%, 5/11) and 19q (27%,3/11) deletions (Raghavan et al., 2003). These data indicate that loss of 1p and19q is a genetic alteration, which is less common in pediatric ODs than in adultones.

In another report, 1p and 19q chromosomal aberrations were found in eightcases of pediatric gliomas, while loss of 1p was found only in one OD sam-ple that derived from a patient with a rapidly progressive disease (Wetzel et al.,2005). Similarly, deletion of 1p was found in only one case of OD in a morerecent study in a cohort of 13 ODs (Kreiger et al., 2005). Because ODs are rel-atively rare in the pediatric population, and 1p and 19q status cannot be used asa molecular diagnostic marker as in the case of adult ODs, they could be con-fused with other types of neoplasms. Other chromosomal abnormalities have beendetected in pediatric ODs. In a study on 145 gliomas, loss of 14q was observedin 8/30 oligodendrogliomas (WHO II), 2/21 oligodendrogliomas (WHO III), 3/9oligoastrocytomas (WHO II), 5/15 oligoastrocytomas (WHO III), 1/7 astrocy-tomas (WHO III), and 11/51 GBM (WHO IV), while no loss was observed in 9astrocytomas (WHO II) (Dichamp et al., 2004). Very little information is avail-able regarding epigenetic dysfunctions in ODs. Evidence from first few studiesis reported below.

9.8.3.1 Aberrant Methylation in Oligodendrogliomas14q32.12. Cytogenetic and molecular studies have shown frequent loss of the14p chromosome arm in human gliomas, and by using differential methylationhybridization in gliomas, Felsberg and coworkers have identified two DNA frag-ments (14q23.1 and 14q32.12) differentially methylated (Felsberg et al., 2006).In a cohort of 43 patients (ranging from 5 to 79 years without other specifica-tions), hypermethylation of the 14q32.12 chromosomal region was found in 2 of5 low-grade oligodendrogliomas, 3 of 6 oligoastrocytomas, and 7 of 12 anaplasticoligoastrocytomas (WHO III), and no aberrant methylation of the same fragmentwas seen in anaplastic oligodendrogliomas, low-grade astrocytomas, AA, andGBM.

Statistical analysis showed that hypermethylation was significantly higher inoligodendroglial-derived tumors compared to that in astrocytomas, indicating thatsilencing of this chromosomal region could be a marker of less aggressive tumors.

CITED4. CITED4 (CREB-binding protein/p300-interacting transactivator withE/D-rich tail 4) is an interacting partner of the acetyltransferases CREB and p300.These two proteins act as transcriptional coactivators, binding to transcription fac-tors and RNA polymerase II (Yuan and Giordano, 2002). CITED4 maps at the1p34.2 chromosomal region, which is deleted in oligodendroglial tumors lackingthe 1p and 19q chromosome arms. The tumor with 1p/19q losses shows enhancedsensitivity to radio- and chemotherapy and favorable prognosis. Recently, differ-entially expressed genes between tumors with or without 1p/19q losses wereinvestigated using microarray-based expression profiling (Tews et al., 2007). In a

PEDIATRIC NEUROECTODERMAL-DERIVED TUMORS 223

cohort of 41 patients ranging from 11 to 91 years with different types of gliomas,CITED4 mRNA were lower in oligodendroglial tumors with 1p and 19q lossescompared to tumors with a normal genetic arrangement. CITED4 gene did notshow genetic mutations, suggesting that epigenetic mechanisms might regulateits expression. In a group of 62 gliomas, CITED4 promoter hypermethylationresulted in 31 of 34 (91%) ODs lacking 1p/19q, whereas only in 2 of 28 ODs(7%) there was no loss of 1p/19q. These results indicate that in ODs hypermethy-lation may be responsible for CITED4 loss of expression, and that this epigeneticabnormality strictly correlates with 1p/19q deletions. CITED4 hypermethylationwas significantly associated with longer recurrence-free and overall survival inpatients with oligodendroglial tumors irrespective of their WHO grade, and couldrepresent a novel prognostic marker in OD patients (Tews et al., 2007).

9.9 PEDIATRIC NEUROECTODERMAL-DERIVED TUMORS

9.9.1 Medulloblastoma

Medulloblastoma (MB) is the most prevalent brain tumor in children youngerthan seven years and the most frequent WHO grade IV malignant brain tumorin childhood while it is rarely seen in adults (Rathi et al., 2003; Lindsey et al.,2004). It originates in the cerebellum and is derived from embryonic cells ofneuroectodermal origin (neuronal precursors) belonging to the family of PNETs.MB consists of a group of clinically and histologically heterogeneous neoplasms,including classic, desmoplastic/nodular, and large cell/anaplastic tumors. Tumordifferentiation grade is associated with clinical outcome, the anaplastic form beingthe less differentiated and the most aggressive. Nevertheless, coexistence withinthe same tumor of areas with a different grade of cell differentiation often makesthe classification difficult. The desmoplastic/nodular variant of MB is the preva-lent form occurring in children younger than three years and has a better outcomethan most MBs in younger patients, whereas the histological anaplastic subtypedevelops in children older than three years and is associated with a poor prognosis(Lindsey et al., 2004). MB cells are highly invasive and often lead to metastasis bydissemination through the cerebrospinal fluid. Usually, five-year survival rates of60–70% are achieved using current risk-adapted combination therapies. Aggres-sive invasive growth of MB requires a strong and multimodal therapy includingneurosurgery, craniospinal irradiation, and chemotherapy. Current treatments areassociated with neurological deficits in psychological development, especially inyounger children (Ebinger et al., 2004; Lindsey et al., 2004). MB is character-ized by a large number of chromosomal rearrangements; in particular, molecularcytogenetic studies have shown that these genetic aberrations modify importantcellular pathways leading to constitutive activation of the Sonic hedgehog andthe Wnt/Wingless signaling pathways, alterations in the TP53 tumor-suppressorpathway, and amplification of the MYC family of oncogenes (Lindsey et al.,2004). In addition, recent data suggest that genetic events are, in many cases,associated with epigenetic modifications of TSGs.


Fraga and coworkers have shown that loss of acetylation of H4K16 was afrequent feature of cancer (Vire et al., 2006). In humans, one of the major proteinsinvolved in H4K16 acetylation is MOF (Cheung et al., 1998), and recent evidencereports the hMOF loss in breast cancer and MB samples (Pfister et al., 2008). Inparticular, in 180 MB patients examined by immunohistochemistry, about 40%displayed loss of acetylation of H4K16 , and in a subset of these patients examinedfor hMOFmRNA expression, 11/14 (79%) showed significant downregulation.Loss of hMOF was found more frequent in aggressive anaplastic and classicthan in desmoplastic MB, thus representing a molecular unfavorable prognosticbiomarker in MB.

Several specific studies demonstrated that the methylation status of more than6% of CpG islands was altered in MB, indicating that normal patterns of DNAmethylation are disrupted. In addition, about 16 of the 24 genes analyzed bymany researchers in at least 16 published studies were hypermethylated at a dif-ferent level in MB (p16/INK4a , p14/ARF , TP73 , TP53 , RB1 , RASSF1A, HIC1 ,EDNRB , CASP8 , DAPK , CDH1 , THBS1 , TIMP3 , GSTP1 , MGMT , and MCJ )(Lindsey et al., 2004).

Genes hypermethylated in MB are involved in principal cellular functionssuch as cell cycle control (TP53, TP73, RB1 , p16/INK4a , p14ARF , microtubulestabilization and regulation of mitosis (RASSF1A), regulation of transcription(HIC1 ), apoptosis (CASP8 ), cell adhesion (CDH1 ), extracellular matrix home-ostasis (TIMP3 ), and DNA repair (MGMT ). Among these, the RASSF1A, CASP8 ,and HIC1 genes show a high percentage (30–60%) of hypermethylation in pri-mary MBs, while the remaining genes show 0–20% hypermethylation. Even ifthe frequencies of methylation can change among individual studies and techni-cal methods used, RASSF1A, CASP8 , and HIC1 are seen to have higher levelsof methylation in MB, compared to normal brain and cerebellar biopsy samples.

An important aspect is the difficulty in correctly comparing the results derivedfrom diverse experimental methods having different sensitivities such as bisul-phite sequencing, COBRA (combined bisulfite restriction analysis), and MSP(Lindsey et al., 2004). Contradictory results are often reported for some genes,possibly depending on these technical approaches, as in the case of p16/INK4aand p14ARF . Two studies showed consistent methylation of p16/INK4a andp14/ARF in two cohorts of patients composed of 11 and 44 primary MBs, lead-ing to hypermethylation rates of 63% (7/11) and 45% (5/11) for the first studyand 39.5% (17/43) and 26.5% (11/42) for the second one, respectively, detectedby MSP (Gonzalez-Gomez et al., 2003a; Muhlisch et al., 2006). It is noteworthythat, when COBRA analysis was additionally used in the second study, the fre-quency of methylation decreased to 26% (6/23) for p16/INK4a and 0% (0/8) forp14ARF , confirming that the sensitivity of the two techniques is diverse. Con-versely, two other research groups reported <7% in 44 and 23 primary MBs forboth genes (Lindsey et al., 2004). Even if many molecular and functional detailsare not yet clear, the three genes RASSF1A, CASP8 , and HIC1 , are consideredprincipal candidate TSGs epigenetically inactivated by hypermethylation in MB.


9.9.1.1 Aberrant Methylation in Medulloblastoma

RASSF1A. The RASSF1A gene is located at 3p21.3 and encodes a protein witha ras-association domain playing a role in ras-dependent apoptosis (Vos andClark, 2005). In Matallanas’s study, the authors demonstrated that RASSF1Aactivation in several tumor cell lines induces YAP1 phosphorylation, nucleartranslocation, and final binding to TP73, resulting in apoptosis (Matallanas et al.,2007). In a large number of solid tumors, such as breast, renal, and nasopha-ryngeal carcinomas and NBs, RASSF1A is altered by hypermethylation on oneallele and genetic mutation of the other allele, and these alterations often corre-late with the tumor stage (Agathanggelou et al., 2003; Armstrong et al., 2007;Michalowski et al., 2008; Rastetter et al., 2007). No RASSF1A mutations havebeen reported in MBs, and about 90% of the most common silencing mecha-nisms to inactivate the RASSF1A gene result in biallelic hypermethylation. ByMSP, Harada and coworkers (Rathi et al., 2003) showed that RASSF1A washypermethylated in about 88% of MBs examined without any significant corre-lation with sex, age, metastasis, and outcome, while high level of methylationwas absent in nonpathological samples. Further, Lusher et al., reported that 79%of primary tumors and 100% of MB cell lines displayed high levels of tumor-specific DNA hypermethylation across the RASSF1A promoter-associated CpGisland (Lindsey et al., 2004). They used COBRA analysis followed by bisulphitesequencing to quantify the extension of methylation in a series of 34 primaryMBs including 27 pediatric and 7 adult cases. A pattern of dense hypermethy-lation was found in 91% (31/34) of tumor samples and in 100% (8/8) of MBcell lines. Importantly, RASSF1A promoter hypermethylation was common toall the histopathological forms and unrelated to the age of the patient. Non-neoplastic cerebral and cerebellar tissue (n = 9) showed no methylation. In asuccessive study, the same group confirmed 93% methylation of RASSF1A in acohort of 28 MB samples, the majority of which was of pediatric origin (Lind-sey et al., 2004). Most frequently, the hypermethylation of CpG islands wascomplete, indicating the involvement of both alleles. Accordingly, Chang et al.found 100% methylation of RASSF1A in primary samples (25/25) and cell lines(3/3) and no methylation in normal brain cerebral and cerebellar tissues (Changet al., 2005).

In the past year, an additional study on PNETs reported elevated methylationof RASSF1A in 19 of 21 (91%) samples and in 3 of 3 (100%) cell lines ofMB by MSP followed by bisulphite sequencing (Inda and Castresana, 2007).Interestingly, in this study, high methylation in all CpG islands of RASSF1Apromoter was observed also in supratentorial PNET (stPNET), a type oftumor with the same neuroectodermal origin as MB but arising in the cerebralhemispheres, suggesting that this molecular abnormality is a signature ofneuroectodermal brain tumors. The biallelic hypermethylation of the RASSF1Apromoter, besides the complete absence of methylation in normal controls,strongly suggests that it is likely to represent a specific feature correlated to thepathogenesis of aggressive MB.


CASP8. The CASP8 gene is located at 2q33-q34 region and encodes the caspase8 cystein protease, which is involved in TRAIL-induced apoptosis. In MBs, theCASP8 gene is reported methylated at the level of CpG islands in both alleleswith high frequencies rather than mutated by a genetic modification. Silenc-ing of the CASP8 gene can induce, as a consequence, the abrogation of theapoptosis pathway favoring cell proliferation and tumor development (Lindseyet al., 2004). For this reason, therapeutic strategies to induce tumor cell deathby TRAIL-signaling activation have been proposed (Eggert et al., 2000). MSPanalysis from Zuzak and colleagues has revealed that more than 75% of CASP8promoter regions in MB cell lines and more than 55% in primary tumors werehypermethylated (Zuzak et al., 2002). More recently, complete methylation wasfound in 15/24 (62%) primary samples of MB by MSP (Gonzalez-Gomez et al.,2003a). It is important to note that in cerebellar normal tissue, hemimethylationcan be usually present, suggesting that further methylation of the other allele canbe an easy event. Further, in these studies unmethylated and methylated areasof CASP8 were found, highlighting the heterogeneous nature of pediatric braintumor tissues. A lower frequency of CASP8 methylation was reported by Lind-sey et al., in a cohort of 44 MB patients (Lindsey et al., 2004). In this study,using MSP and COBRA analysis, 38.6% of 44 tumors (17/44) presented completemethylation, whereas normal cerebellar samples were only partially methylated.

HIC1. HIC1 encodes a zinc finger transcription factor involved in the repressionof specific target genes; even if many molecular details are not known, micewith one disrupted copy of HIC1 develop tumors. The HIC1 gene is located at17p13.3, a region frequently involved in genetic and epigenetic modifications inmany types of cancers, which shows hemimethylation in the normal cerebellum.In MB, it is silenced by deletion events and by aberrant hypermethylation. Studiesby Rood et al. showed that MBs exhibit a pattern of aberrant methylation in theregion of HIC1 and that this is correlated with a poor outcome, suggestingthe functional importance of HIC1 in the aggressiveness of tumors (Rood etal., 2002). Accordingly, Waha et al. demonstrated that HIC1 is frequently andaberrantly hypermethylated in a large number of MB samples; in particular, about85% of MB biopsies and 88% of MB cell lines showed hypermethylation of HIC1(Waha et al., 2003). Finally, HIC1 appeared totally methylated in 17/44 (38.6%)and partially methylated in 26/27 (96%) MB samples (Lindsey et al., 2004).

S100. The family of S100 proteins includes 20 members; among these, 16(S100A1-S100A16) map in 1q21.3 and span a region of 1.65 Mb (Donato,2003; Marenholz et al., 2004). They are involved in multiple processes suchas transcription, cell cycle regulation, cell growth, differentiation, and motility(Marenholz et al., 2004). S100 proteins show tissue-specific expression that isimpaired in a range of diseases (Heizmann et al., 2002; Marenholz et al., 2004;Heizmann, 2005). Deregulation of S100 protein expression and function seemsto be involved in tumor progression. Upregulation of S100A4 was reported formany types of cancers, including MB (Arumugam et al., 2005; Wang et al., 2006),


and hypermethylation of S100A2 and S100A46 promoters has been observed inprostate cancer (Rehman et al., 2005). Recently, the epigenetic regulation ofS100A2 , S100A4 , S100A6 , and S100A10 was investigated in a cohort of 41 MBpatients (1.3–19 years of age) and 6 normal cerebella by bisulphite sequencing(Lindsey et al., 2004). S100A6 was generally unmethylated in normal cerebellumand methylated in 5/40 (12%) patients and 7/9 (78%) cell lines, hypermethyla-tion in patients being significantly associated with aggressive large cell/anaplasticmorphophenotype. S100A10 was unmethylated in control samples, whereas 4 of35 (11.4%) tumors and 7 of 9 (78%) cell lines displayed methylation to a dif-ferent extent. S100A2 showed a methylation pattern that was variable in bothcontrol and MB samples. In contrast, S100A4 was methylated in all nonneoplas-tic tissues, remaining undermethylated in 7/41 (17%) MB and 3/9 (33%) celllines. This finding was consistent with the S100A4 transcript expression beinghigher in MBs than in controls, suggesting a protumorigenic role for this protein.

COL1A2. The COL1A2 gene encodes for the α2 subunit of type I collagen thatis a major component of blood-vessel-associated basement membranes and rep-resents a major fibrillar component of the stroma of most solid malignancies(Tripathi Bhar et al., 2003; Chiba et al., 2005). COL1A2 is a downstream targetof the epidermal growth factor (EGF) signaling, and can suppress cell transfor-mation induced by ras and other oncogenes (Travers et al., 1996; Andreu et al.,1998). It was shown to be upregulated in gastric cancer (Ren et al., 2004) andaberrant methylated in hepatoblastoma and colorectal carcinoma tissue and celllines (Tripathi Bhar et al., 2003; Chiba et al., 2005). COL1A2 mRNA was over-expressed in PNETs, and an increased deposition of the type 1 collagen protein inextracellular matrix was shown by immunohistochemistry on the same samples(Liang et al., 2008). In a cohort of 60 MBs, COL1A2 gene promoter methy-lation was found in 36 of 42 (86%) nondesmoplastic and in 10 of 18 (56%)desmoplastic/nodular tumors with a significant association between methylationand histopathological morphophenotype. In addition, a correlation between ageand COL1A2 methylation was reported. In particular, COL1A2 methylation wasdetected in 40 of 46 (87%) patients older than three years, while it was detectedonly in 6 of 14 (43%) patients younger than three years (Lindsey et al., 2004).Although these findings suggest that desmoplastic/nodular tumors may have dif-ferent molecular features in infants compared to older children, the COL1A2function in MB development requires further investigations.

SGNE1/7B2. SGNE1 /7B2 (secretory granule neuroendocrine protein 1) is thehuman homolog of the murine 7B2 gene both encoding, a Ca2+-dependent serineprotease that shares homology with the endopeptidases subtilisin (in bacteria)and kexin (in yeast). It maps at 15q11–15 chromosome region (Mattei et al.,1990), that is altered in patients with Prader–Willi syndrome (Cassidy, 1997).SGNE1/7B2 acts as a specific chaperone for the proprotein of convertase-2 (PC2)and is selectively expressed in the CNS and in endocrine tissues (Hsi et al.,1982; Iguchi et al., 1984). SGNE1/7B2 and PC2 were coinduced and processed


during neuronal differentiation of P19 cells (Jeannotte et al., 1997). AlteredSGNE1/7B2 expression was found in many types of human tumors [reviewed inMbikay et al. (2001)]. Recently, epigenetic regulation of SGNE7/B2 expressionwas investigated by bisulphite sequencing and COBRA analysis in human MBtissues and cell lines. In a cohort of 23 MBs, consisting of 15 classic and 8desmoplastic cases, hypermethylation of all sites of 5′UTR was found in 12/15(80%) and 4/8 (50%), respectively, whereas the normal fetal cerebellum showeda complete lack of methylation. In addition, hypermethylation was also found in8/8 MB cell lines (100%). Analysis of SGNE1 /7B2 expression in 14 classic and12 desmoplastic MBs in 8 cell lines and 6 fetal cerebellum samples showed lowermRNA expression in tumors than in nonneoplastic tissues, with the exception of1 tumor sample. Treatment with 5-aza-dC of the cell lines resulted in a significantincrease in SGNE1/7B2 expression (Waha et al., 2007). Enforced expression ofSGNE1/7B2 in MB cell lines resulted in inhibition of cell growth and colonyformation, suggesting that this protein could have an antitumor role in MBs.

DKK1. DKK1 (Dickkopf-1) is a protein that antagonizes the WNT signaling,an important pathway that controls development and homeostasis. WNT pro-teins are soluble factors that, after interaction with their receptors Frizzled andLRP5/6, favor β-catenin-dependent transcriptional activation of genes involvedin proliferation such as, cyclin D1 , AXIN2 , and the oncogene c-Myc. It hasbeen observed that DKK1 expression is downregulated in human colon cancers(Gonzalez-Sancho et al., 2005), and that its downregulation seems to depend onhypermethylation of the gene promoter (Garcia et al., 2002), as reported for otherhuman cancer cell lines (Lee et al., 2008). To date, no significant data regardingaberrant methylation of DKK1 have been reported in MB. Nevertheless, a recentstudy has identified the DKK1 gene as significantly upregulated after HDACinhibition in MB (Vibhakar et al., 2007). Indeed, treatment with the HDACi thri-costatin (TSA) was sufficient to restore DKK1 expression in MB cell lines. Bychromatin immunoprecipitation assay (ChIP), Vibhakar and colleagues reportedthat in a cohort of 10 pediatric MBs (<18 years of age) DKK1 expression was80% lower than in normal cerebellum tissues. Altogether, these findings supportthe importance of histone acetylation in regulating DKK1 gene expression andimplicate that the aberrant deacetylation-dependent transcriptional silencing ofthis gene is a potential component of MB pathogenesis.

9.9.2 Supratentorial Primitive Neuroectodermal Tumors (stPNETs)

The stPNETs belong to the CNS neuroectodermal tumor family and, with MB,constitute the most common malignant embryonal brain tumors in children. Theyarise in the cerebrum or pineal body and are relatively undifferentiated andextremely aggressive (WHO grade IV). Moreover, even if they share histopatho-logical features with MB, they clearly differ in specific cytogenetic abnormalities(Russo et al., 1999). While MBs are responsible for 20–25% intracranial malig-nancies of childhood, stPNETs represent only 3–7% of pediatric CNS tumors


and are more common in young adults (21–40 years of age). In some clinicalstudies, it has been shown that stPNETs can also develop as secondary malignan-cies after radiation therapy (Ha et al., 2006). In any case, the five-year survivalrate of children with stPNET is 20–30% being lower than that for children withMB.

Epigenetic studies on stPNET indicated methylation of some genes alreadyshown methylated in MB but also methylation in loci not affected in MB. How-ever, because stPNET is a very rare tumor, molecular studies have been difficultand results sometimes unclear.

In two reports, RASSF1A showed a pattern compatible with biallelic hyperme-thylation in 67% (6/9) and 90% (19/21) primary pediatric stPNETs, in agreementwith data on MB (Chang et al., 2005; Muhlisch et al., 2006). In both studies,aberrant methylation of the RASSF1A promoter was specific of stPNET samplesbeing absent in normal brain samples. In particular, Muhlisch et al., through semi-quantitative MSP, demonstrated that 47% of pediatric patients (10/21) showed80–100% of hypermethylation frequency of RASSF1A promoter, 29% (6/21) had35–70% and only 19% (4/21) presented a frequency <5%. The extent of methy-lation was confirmed by bisulfite sequencing. These results were corroborated byRT-PCR showing high expression in normal brain tissue from infants and chil-dren and weak or absent expression in two stPNETs. Finally, in a recent study,hypermethylation of RASSF1A was observed by MSP in 2/2 stPNET, one froma pediatric and the other from an adult patient, and in five stPNETs cell lines(Inda and Castresana, 2007). Accordingly, no RASSF1A expression was detectedin cell lines. Taken together, these results indicate that epigenetic inactivation ofRASSF1A may play a role in the development of stPNET, as already hypothesizedfor MB.

On the contrary, CASP8 , which has been demonstrated consistently methylatedin MB, showed no relevant differences in methylation frequency between stP-NET tissue and control cerebral matter (Muhlisch et al., 2006). In one report, byMSP and COBRA analysis no methylation was seen for p16/INK4a , p15/INK4b,p14ARF, SOCS1, DUTT1 , and DAPK1 promoters in five pediatric stPNET sam-ples (Muhlisch et al., 2007). In addition, CDH1 and TIMP3 were found unmethy-lated or <10% methylated.

MCJ (DNAJD1 ) is a member of the DNAJ family of cochaperone proteins.Even if their cellular functions have not yet been well characterized, membersof the J-domain superfamily are involved in many biological mechanisms, suchas regulation of protein kinases, protein folding, protein translocation, and cellcycle control by DNA tumor viruses (Lindsey et al., 2004).

Loss of MCJ expression confers resistance to chemotherapeutic agents usedin the treatment of ovarian cancer; in particular, MCJ is epigenetically silencedby promoter methylation in ovarian cancer patients and it can be associatedwith both response to chemotherapy and to OS. Lindsey and colleagues investi-gated the methylation status of MCJ promoter in 10 pediatric stPNET and foundmethylation in 3 samples (30%). By MSP, COBRA, and bisulphite sequencing,they showed extensive methylation in one sample, whereas in two samples this


was limited. In any case, no correlation with the histological subtypes or age atdiagnosis was present.

Methylation of the MCJ CpG island was not found in normal nonneoplasticbrain tissues, indicating that MCJ methylation was a tumor-specific event, whicharises de novo in stPNET (Lindsey et al., 2004).

Acknowledgment

This chapter was supported by a grant from the Italian Ministry of Health andWelfare.

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Cancer Epigenetics (Biomolecular Therapeutics for Human Cancer) || Epigenetics in Pediatric Cancers