Identification of tumour-specific epigenetic events in medulloblastoma development by hypermethylation profiling

Janet C. Lindsey1,*, Meryl E. Lusher1,*, Jennifer A. Anderton1, Simon Bailey2, Richard J. Gilbertson3, Andrew D.J. Pearson1, David W. Ellison1 and Steven C. Clifford1,4

1 Northern Institute for Cancer Research, The Medical School, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, UK, 2 Sir James Spence Institute of Child Health, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne NE1 4LP, UK and 3 Department of Developmental Neurobiology, St Jude Children's Research Hospital, 332 N. Lauderdale Street, Memphis, TN 38105, USA

4 To whom correspondence should be addressed Email: s.c.clifford{at}ncl.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Medulloblastoma arises in the cerebellum and is the most common malignant brain tumour of childhood, however its molecular basis is not well understood. To assess the role of aberrant epigenetic events in medulloblastoma and identify critical genes in its development, we profiled the promoter methylation status of 11 candidate tumour-suppressor genes (TSGs; p14ARF, p15INK4b, p16INK4a, CASP8, HIC1, EDNRB, TIMP3, TP73, TSLC1, RIZ1 and RASSF1A) in medulloblastoma cell lines, primary tumours and the normal cerebellum. Gene-specific TSG methylation was a significant feature of both medulloblastomas and the cerebellum. Extensive hypermethylation of RASSF1A was detected frequently in medulloblastomas but not in the normal cerebellum (41/44 primary tumours versus 0/5 normal cerebella). In contrast, complete methylation of HIC1 and CASP8 in a subset of primary tumours (17/44 and 14/39) occurred against a consistent background of partial methylation in the normal cerebellum. These data therefore indicate that extensive methylation of RASSF1A, HIC1 and CASP8 are tumour-specific events in medulloblastoma. Moreover, methylation of these genes in medulloblastoma cell lines was associated with their epigenetic transcriptional silencing and methylation-dependent re-expression following treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. The remaining genes studied showed either low frequency methylation (p14ARF, p16INK4a, RIZ1; <7% of cases), no evidence of methylation (p15INK4b, TIMP3, TP73, TSLC1), or comparable patterns of methylation in the normal cerebellum (EDNRB), suggesting that their hypermethylation does not play a major role in medulloblastoma. Our data demonstrate that tumour-specific hypermethylation affects only a subset of genes, and does not support the existence of a concordant methylation phenotype in this disease. We conclude that epigenetic TSG inactivation is a significant feature of medulloblastoma, and identify RASSF1A, HIC1 and CASP8 as potentially critical genes in its pathogenesis. Furthermore, methylation observed in the normal cerebellum emphasises the requirement for appropriate control tissues when assessing the tumour-specificity of TSG hypermethylation.

Abbreviations: 5-aza CdR, 5-aza-2'-deoxycytidine; COBRA, combined bisulphite and restriction analysis; MSP, methylation-specific PCR; TSG, tumour-suppressor gene


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Medulloblastoma, the most common malignant brain tumour of childhood, is a primitive neuro-ectodermal tumour arising in the cerebellum. The aggressive clinical behaviour of the tumour and the cognitive and endocrinological long-term side effects of current therapies make both the development of prognostic indicators for disease stratification and the identification of new therapeutic targets a major goal. Current understanding of the molecular biology of medulloblastoma is limited. Cytogenetic studies have described consistent chromosomal aberrations, however molecular genetic studies have identified specific genetic abnormalities in only a small proportion of tumours (reviewed in refs 1,2).

Hypermethylation of promoter-associated CpG islands leading to transcriptional silencing has emerged as an important mechanism of epigenetic inactivation of tumour suppressor genes (TSGs) in cancer development (reviewed in ref. 3). Using a genome-scanning approach, aberrant patterns of CpG island methylation have been described in medulloblastoma (4,5), although few gene-specific events have been identified to date. Recently, we have demonstrated bi-allelic epigenetic inactivation by promoter methylation of the tumour suppressor gene RASSF1A (ras-association domain family protein 1, isoform A) in medulloblastoma (6), further highlighting the potential significance of epigenetic TSG inactivation in the development of this tumour.

To examine more widely the prevalence and role of aberrant promoter methylation events in medulloblastoma and to identify further tumour-specific events in its pathogenesis, we have determined the methylation status of the promoter-associated CpG islands of a series of known or candidate TSGs in primary medulloblastoma tumours and the normal cerebellum. In addition to RASSF1A, three groups of candidate genes were selected for analysis: (i) genes which have been reported previously to show evidence of methylation in medulloblastoma [CASP8 (Caspase 8, cysteine-aspartic acid protease 8), HIC1 (hypermethylated in cancer 1) and p16INK4a] (5,810), (ii) genes which are epigenetically inactivated in other brain tumours [p14ARF p15INK4b, TIMP3 (tissue inhibitor of metalloprotease 3) and TP73] (1115) and (iii) genes which exhibit methylation in several different cancer types, suggesting frequent involvement in tumour development [EDNRB (endothelin B receptor), TSLC1 (tumour suppressor gene in human lung cancer) and RIZ1 (retinoblastoma protein interacting zinc finger gene)] (7,1620).

This parallel examination of multiple promoter-associated CpG islands has allowed the compilation of an extensive profile of gene-specific methylation events in medulloblastoma. Importantly, the analysis of normal cerebellar tissue has permitted the identification of tumour-specific hypermethylation of a subset of genes, which we show is associated with their transcriptional silencing. This highlights the potential importance of these epigenetic events in medulloblastoma pathogenesis. Finally, we make a preliminary assessment of the clinico-pathological significance of tumour-specific methylation in medulloblastoma.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines and patient material
Eleven medulloblastoma cell lines (DAOY, D283 Med, MHH-MED-1, MHH-MED-8A, D341 Med, D384 Med, D425 Med, D458 Med, UW402 and UW228) were studied. D425 Med and D458 Med were derived from the primary and a metastatic tumour from a single patient, respectively; UW228-2 and UW228-3 were derived from the same tumour but exhibit phenotypic differences (21), all other cell lines were derived independently. All cells were grown under recommended culture conditions, and cell line identity was confirmed prior to use by karyotyping (data not shown). Cell line DNA was extracted using the Qiagen DNeasy kit (Qiagen, Crawley, UK).

A cohort of 44 primary medulloblastomas were analyzed, representative of all the major histopathological subtypes (25 classic, seven large cell/anaplastic and 12 nodular/desmoplastic tumours), and adult and paediatric patients (10 infants <3 years, 30 children 3–16 years and four adults >16 years). Clinical and pathological data were centrally reviewed for this study. The presence of metastases was detected on a pre-operative MRI scan. DNA was extracted from frozen tissues using standard methods and from formalin-fixed, paraffin-embedded tissue using a Nucleon hard tissue kit (Amersham Biosciences, Little Chalfont, UK). Normal cerebellar control DNA consisted of post-mortem material from two infants (new born and 25 months) and three adults (60, 67 and 68 years) who had died of non-neoplastic conditions. Normal peripheral blood DNA consisted of a pool of 20 newborn cord blood DNAs. Local Ethical Committee and Institutional Review Board approval has been obtained for the collection, storage and biological study of all material.

Analysis of promoter methylation status
Bisulphite treatment of DNA was carried out using a CpG genome DNA modification kit (Serologicals, Livingston, UK) according to the manufacturer's instructions. The methylation status of the CpG islands of p14ARF, p15INK4b, p16INK4a, CASP8, HIC1, EDNRB, TIMP3 and RIZ1 was determined by methylation-specific PCR (MSP) (22) using previously published primer sets (13,16,19,2225). The methylation status of the CpG islands of RASSF1A, TP73 and TSLC1 was determined by direct sequencing of the reverse strand and estimation of the relative peak heights of PCR products generated from bisulphite treated DNA using previously published primers (14,17,26). The unmethylated control for MSP consisted of pooled normal neonatal cord blood DNA (see above), the methylated control was the same sample methylated in vitro by SssI methylase (New England Biolabs, Hitchin, UK).

Thirty nanograms of bisulphite treated DNA was used per reaction, the PCR reactions were carried out using previously published conditions. MSP PCR products were separated on a 2.5% agarose gel in 1x TBE [0.09 M Tris–Borate, 0.002 M EDTA (pH 9)]. Products analysed by sequencing were directly sequenced with a CEQ DTCS kit (Beckman Coulter, High Wycombe, UK) using the antisense primer to obtain the reverse sequence. Sequenced products were analyzed on a CEQ 2000XL DNA analysis system (Beckman Coulter), and the methylation status at each CpG residue determined by assessment of the relative peak intensities.

COBRA (combined bisulphite and restriction analysis) of the CASP8 promoter was performed by overnight digestion of a PCR product generated using a non-differential primer set CASP8SQ2 (9) with Taq1 (MBI Fermentas, Hanover, MD) at 65°C, which cuts once within the 455 bp PCR product generating products of 396 and 59 bp. The 455 bp uncut product and the 396 bp digested product were separated on a 4% Nusieve 3:1 agarose gel in 1x TBE.

5-Aza-2'-deoxycytidine (5-aza CdR) treatment and RT–PCR
Four cell lines (D425 Med, MED-8A, DAOY and D283 Med) were grown in the presence or absence of the demethylating agent 5-Aza CdR (5 µM) for 4 days. Medium was renewed daily. HeLa cells were also grown as a positive control as these expressed all four transcripts. RNA was extracted from 107 cells using an RNeasy kit (Qiagen). RNA concentration was determined by spectrophotometry, and agarose gel electrophoresis was used to confirm consistent RNA integrity and quantity from each cell line. One microgram of total RNA was used to synthesize cDNA using a reverse transcription system (Promega, Southampton, UK). cDNA was synthesized using random primers and oligo-d(T) primers in separate reactions, followed by pooling. Equivalent amounts of this cDNA was used for PCR amplification of the RASSF1A, CASP8, HIC1 and ß-actin transcripts. The primers for detecting RASSF1A expression were as described previously (26) from exons 2aß and exon4, generating a 242 bp product. CASP8 primers were a previously published set from exons 1 and 3 generating a 379 bp product (9). HIC1 primers were designed for exon 1B 5'-GCGCGGCGGGGGCTGAGAC and exon 2 5'-GCCCTTGGTGCGCTGGTTGTTGAG, product size 244 bp, to amplify the major transcript (28). ß-Actin, a housekeeping gene, was used as a control for RNA integrity using primers BA67 and BA68 (29). Annealing temperatures for RASSF1A, CASP8, HIC1 and ß-actin were 60, 57, 66 and 60°C respectively. ß-Actin PCRs were amplified for 25 cycles, all other reactions were amplified for 35 cycles. PCR products were electrophoresed on a 2.5% agarose gel in 1x TBE.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methylation status of CpG islands in medulloblastoma cell lines
In order to highlight genes that are potentially methylated in medulloblastoma tumours, the methylation status of the 11 candidate genes (RASSF1A, CASP8, HIC1, p16INK4a, p14ARF, p15INK4b, TIMP3, TP73, EDNRB, TSLC1 and RIZ1) was initially investigated in a panel of 11 medulloblastoma cell lines by MSP and bisulphite sequencing, using previously validated primer sets and conditions (13,14,16,17,19,2226). A summary of these results is presented in Table I. All medulloblastoma cell lines showed evidence of methylation of multiple genes, with between two and six of the 11 CpG islands examined methylated in each cell line.


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Table I. Methylation status of 11 tumour-related genes in a panel of eleven medulloblastoma cell lines

 


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Fig. 1. (A) Methylation status of genes in medulloblastoma tumours and the normal cerebellum. Representative examples of MSP results for CASP8, HIC1, EDNRB and p14ARF are shown for tumours, normal cerebella and controls. Controls consisted of fully methylated DNA, fully unmethylated DNA and mixtures of the two in the specified ratios to confirm that the two PCR reactions were of approximately equal efficiency and capable of detecting the minority species of DNA at these concentrations. These controls were performed for all MSP reactions. (B) Confirmation of CASP8 CpG island methylation by COBRA analysis. Methylation status of the CASP8 CpG island determined by Taq1 digestion, which cuts the 455 bp PCR product once at a methylated CpG site generating products of 396 and 59 bp (not shown). The ratio of the uncut (455 bp) product to the cut (396 bp) product therefore approximately reflects the ratio of unmethylated to methylated DNA present in the sample. The methylation status determined by MSP result is also shown for comparison.

 
Tumour-related genes RASSF1A and HIC1 were most extensively methylated, with evidence of methylation in all cell lines and complete methylation in 10/11 and 9/11 cell lines, respectively. CASP8 and EDNRB also exhibited evidence of methylation in a high proportion of cell lines (8/11 and 9/11, respectively), with instances of both full and partial methylation observed. A smaller proportion of cell lines were methylated for p14ARF (4/11), p16INK4a (1/11) and RIZ1 (2/11). Methylation of p14ARF was partial when present, whereas p16INK4a and RIZ1 each showed complete methylation in a single cell line. Genes p15INK4b, TP73, TSLC1 and TIMP3 showed no evidence of methylation in any cell line, and it was therefore concluded that epigenetic inactivation of these genes was unlikely to play a significant role in medulloblastoma development and they were not investigated further.

Methylation status of tumour-related genes in primary medulloblastoma tumours and the normal cerebellum
All genes showing evidence of methylation in cell lines were further analysed in a representative panel of 44 primary medulloblastoma tumours (see Figure 1A and Table II). RASSF1A, CASP8, HIC1 and EDNRB showed evidence of partial or complete methylation in a large percentage of tumours (93, 97, 98 and 54% of analysable samples, respectively), demonstrating that gene-specific promoter methylation is a feature of medulloblastomas. Evidence for total methylation of RASSF1A, CASP8 and HIC1 was found in a subset of tumours (57, 36 and 39% of analysable samples, respectively), whereas EDNRB showed evidence of only partial methylation. p14ARF and p16INK4a showed partial methylation in a small percentage of tumours (4.5 and 7%, respectively). No evidence of RIZ1 promoter methylation was detected in any tumour (data not shown), suggesting that RIZ1 methylation in medulloblastoma either is very rare or a cell line-specific phenomenon.


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Table II. Methylation status of tumour-related genes in primary medulloblastoma tumours and normal cerebellar samples

 
In order to assess whether the methylation patterns detected in primary medulloblastomas were tumour-specific or reflective of any background level of tissue-specific methylation in their tissue of origin (i.e. cerebellum), genes showing evidence of methylation in tumours were also analysed in five normal cerebellar DNA samples [two infant (newborn and 25 months) and three adult (aged 60, 67 and 68 years) (see Figure 1A and Table II)].

RASSF1A showed no evidence of methylation in the normal cerebellum, in contrast to frequent methylation in tumours. Our previous study (6) examined RASSF1A promoter methylation status in 34 tumours. This study has determined RASSF1A status in a further 28 tumours bringing the total number analysed to 62, of which 54 (87%) are methylated. p14ARF and p16INK4a similarly showed no evidence of methylation in the normal cerebellum, compared with partial methylation in a small percentage of medulloblastomas, however, to be certain this methylation is tumour-specific and not a rare non-tumour-related event, a comparable number of normal cerebella should be examined. These data further demonstrate that hypermethylation of the RASSF1A CpG island is a frequent and tumour-specific event in medulloblastoma development with a potentially significant role in its pathogenesis, and suggest that p14ARF and p16INK4a methylation may also contribute to tumour formation in a small subset of cases.

EDNRB showed evidence of partial methylation in both tumour samples and normal cerebella. The percentage of methylated samples observed was similar [3/5 (60%) of normal cerebella and 26/44 (59%) of tumours], the relative amounts of unmethylated and methylated products seemed comparable, and methylation status appeared independent of age. It was therefore concluded that the EDNRB methylation observed in tumours is reflective of a normal level of tissue-specific methylation, and is not a tumour-related event.

CASP8 showed consistent evidence of partial methylation in all normal cerebellar DNA samples analysed, however in contrast a proportion of medulloblastoma samples (14/39 or 36%) displayed complete methylation of this region by MSP analysis. Twenty-four of the 25 remaining tumour samples displayed a partial methylation status comparable with that observed in the normal cerebellum. To investigate these results further, PCRs of the CASP8 CpG region were performed using primers capable of amplifying both methylated and unmethylated DNA sequences (i.e. containing no CpG sites), followed by a restriction digest (Taq1) to discriminate between methylated and unmethylated products (COBRA). This analysis provides a more accurate reflection of the relative proportions of methylated and unmethylated product. Results obtained (Figure 1B) confirm data from the MSP analysis; cerebellar DNA shows a degree of CASP8 methylation with the unmethylated band predominating in these samples, whereas in medulloblastoma DNAs the methylated band predominates. Tumour samples categorized as fully methylated by MSP analysis had a higher ratio of methylated to unmethylated product than those categorized as partially methylated. A small amount of unmethylated DNA product could be seen in samples classed as fully methylated by MSP analysis, which may reflect either increased sensitivity of the COBRA analysis or differences in methylation at individual CpG sites. These data indicate that an enhanced tumour-related methylation status is observed for CASP8 in a proportion of medulloblastomas, which occurs against a background of tissue-specific partial methylation in the normal cerebellum.

HIC1 showed an equivalent pattern of methylation to CASP8, similarly indicative of tumour-specific alterations to its methylation status. Consistent evidence of partial HIC1 methylation was observed in all normal cerebellar samples analysed, whereas complete methylation was detected in a significant proportion of tumours (17/44 or 39%). Patterns of partial methylation reflective of the normal cerebellum were observed in 26 of the 27 remaining tumour samples.

Overall, these analyses provide evidence that gene-specific DNA methylation is a significant feature in the normal cerebellum, which must be considered in the assessment of tumour-specific methylation states. Notably, this is illustrated by our observation that the three genes that showed evidence of methylation in normal cerebellar material (CASP8, HIC1 and EDNRB) did not exhibit methylation in normal blood DNA samples (see controls, Figure 1A), highlighting the importance of comparing methylation status in tumours against relevant control tissues, as opposed to a general control.

Assessment of clinicopathological correlations and patterns of concordant methylation
RASSF1A methylation and complete methylation of HIC1 or CASP8 were defined as tumour-specific events. Methylation of RASSF1A was very frequent in our cohort (41/44 tumours) and occurred in tumours of all clinical and pathological sub-groups assessed. In view of this, examination of RASSF1A methylation in a much larger cohort will be required to assess any relationship to clinical and pathological features. Using data from our cohort, a preliminary assessment was made of any potential relationships between full methylation of CASP8 and HIC1 and clinical and pathological disease features [age at diagnosis (by Mann–Whitney and t-tests), sex, metastatic stage and pathological sub-type (using {chi}2 and Fisher's exact tests)]. Clinical data is summarized in Table II. Tumour-specific methylation of HIC1 was detected in tumours of all clinical and pathological sub-groups assessed, and no relationship to patient age, sex, metastatic status or histology was observed in this study. However, while complete CASP8 methylation was commonly observed in tumours of classic and large cell/anaplastic pathology (14/27 cases), it was not a feature of nodular/desmoplastic cases (0/11, P = 0.002 by Fisher's exact test) (see Table II). No other significant correlations with clinical or histopathological criteria were seen.

We also assessed whether there was any evidence of concordant tumour-specific methylation of multiple tumour-related genes in medulloblastomas. No evidence was found to suggest the presence of concordant methylation, and no statistically significant associations were observed between the methylation status of any two individual genes (by Fisher's exact test, see Table II). These findings suggest that a global CpG island methylator phenotype (CIMP) does not exist in this disease.

Functional significance of RASSF1A, CASP8 and HIC1 methylation in medulloblastoma cell lines
The analysis of CpG island methylation status has identified RASSF1A, CASP8 and HIC1 as genes frequently methylated in medulloblastoma and whose methylation was differential between tumour samples and normal cerebella. To investigate whether the observed methylation is associated with transcriptional silencing of these genes, expression of RASSF1A, CASP8 and HIC1 was examined by RT–PCR in four medulloblastoma cell lines, before and after treatment with the demethylating agent 5-aza-CdR. The results are shown in Figure 2.



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Fig. 2. Reverse transcription–PCR analysis of RASSF1A, CASP8 and HIC1 gene expression levels in four medulloblastoma cell lines with (+) and without (–) treatment with 5 µM 5-Aza CdR. HeLa cells were used as a positive control as these expressed all three transcripts. ß-Actin was used as a control for RNA integrity and quantity. Results were reproducible over replicate treatments (data not shown). The methylation status of each cell line is also shown (see Table I for definitions).

 
RASSF1A methylation was strongly associated with its transcriptional silencing (absent or reduced gene expression), and restoration of expression following 5-aza-CdR treatment, in all four lines tested. This confirms and extends our previous work (6) and also serves as a positive control for our other analyses, confirming that 5-aza-Cdr treatment was successful.

For CASP8, the unmethylated cell line DAOY showed clear expression of the gene transcript before 5-aza-CdR treatment, whereas in contrast expression in the methylated cell lines was reduced (D283, MED8A) or absent (D425) prior to treatment, and increased following treatment. For HIC1, the partially methylated cell lines DAOY and MED8A showed higher pre-treatment expression of the HIC1 transcript than fully methylated cell lines (D283 and D425). Both fully methylated lines showed evidence of absent or reduced HIC1 expression, which was increased following treatment. The CpG islands for CASP8 and HIC1 are less clearly defined than that for RASSF1A. The region assessed by MSP for the CASP8 gene only partially fulfils the criteria for a CpG island (9) and the entire HIC1 gene lies within a CpG rich region. A more detailed analysis of the status of individual CpG sites, and expression of any alternative gene transcripts, will therefore be required to fully establish the relationship between methylation patterns and gene expression for these genes. Nevertheless, methylation of the MSP assessed CpG sites for CASP8 and HIC1 was associated with gene expression status in these lines, although the relationship was less pronounced than that between RASSF1A CpG island methylation and expression. These data indicate that the tumour-specific methylation states we have observed for RASSF1A, CASP8 and HIC1 in medulloblastomas are associated with their transcriptional silencing, and suggest that methylation of these genes is of potential functional significance in medulloblastoma development.


    Discussion
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 Abstract
 Introduction
 Materials and methods
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Inactivation of TSGs by promoter hypermethylation has recently emerged as an important mechanism in tumourigenesis (3). Genes that are hypermethylated in tumours relative to normal tissue may have potential as diagnostic markers or prognostic indicators, and the functional pathways in which they reside may represent potential targets for therapeutic exploitation. This study profiles the CpG island methylation status of 11 tumour-related genes in medulloblastoma cell lines, primary tumour samples and the normal cerebellum, in order to assess whether promoter hypermethylation is a significant event in medulloblastoma development, and to identify critical gene-specific events in its pathogenesis.

The results show that CpG island methylation is a frequent and gene-specific phenomenon in medulloblastoma cell lines and tumours, with methylation of at least one CpG island occurring in all tumour samples and at least two islands in medulloblastoma cell lines. Methylation in tumours was limited to a subset of six of the 11 genes (RASSF1A, CASP8, HIC1, p14ARF, p16INK4a and EDNRB). A frequent role for epigenetic inactivation of p15INK4b, TP73, TSLC1 and TIMP3 was discounted on the basis of no evidence of methylation in cell lines, which typically show higher levels of CpG island methylation than primary tumours (30,31) and for epigenetic inactivation of RIZ1, which showed no evidence of methylation in primary tumours.

Based on the CpG islands analysed in this study, we have identified gene and tissue-specific methylation as a significant phenomenon in the normal cerebellum (observed for EDNRB, HIC1 and CASP8). For HIC1 and CASP8, this methylation was consistently observed and independent of age, indicating that our findings represent a basal level of tissue-specific methylation and not an ageing-related event. The underlying nature, pattern and significance of the partial methylation observed in the normal cerebellum are unclear. Our results do not necessarily support a global mono-allelic methylation of these genes in this tissue. The cerebellum is made up of different cell types and it is possible that in certain cell types the promoter is methylated in this manner, and this now requires further clarification. Of particular note, the observation that the vast majority of medulloblastomas show a degree of methylation of HIC1 and CASP8 (43/44 and 38/39 of analysable samples, respectively), suggests that medulloblastomas originate from a cell type within the cerebellum with a basal level of methylation of these genes.

The comparison of methylation states in tumours with normal cerebella has enabled tumour-specific methylation, which may be relevant to tumour development to be distinguished from tissue-specific background methylation. The presence of such methylation in non-neoplastic tissue highlights the importance of comparing tumours with matched control tissue and the limitations in studies, which use DNA from an unmatched normal tissue (e.g. blood) as a control. From a comparison of primary tumour samples and normal cerebellar samples, genes methylated in medulloblastoma tumours can be divided into four different categories: (i) those frequently methylated in a wholly tumour-specific manner (RASSF1A), (ii) infrequently methylated genes for which methylation appears to be tumour-specific (p14ARF and p16INK4a), (iii) those where frequent methylation reflects the background methylation of the tissue the tumour is derived from (EDNRB) and (iv) a class of genes that shows an increased (complete) methylation status in tumours, which occurs against a background of partial methylation in the normal cerebellum (CASP8 and HIC1). Statistical analysis showed that genes were methylated independently of one another and do not support the existence of a ‘CpG hypermethylator’ phenotype (32) affecting multiple genes in this tumour type.

We have shown previously that the promoter of RASSF1A is extensively methylated in medulloblastoma tumours (6). This study confirms that RASSF1A is frequently methylated (in 93% of this cohort), and represents the most common gene-specific defect reported to date in this disease. Methylation occurs in all histopathological and clinical disease subtypes. Importantly, our current data extend these findings to show that this methylation is not present in the normal cerebellum, demonstrating that RASSF1A is methylated in a tumour-specific manner during medulloblastoma development. Methylation of the RASSF1A promoter was clearly associated with transcriptional silencing in all medulloblastoma cell lines examined. Whilst the RASSF1A protein product has been demonstrated to have tumour suppressor properties in vitro in several different adult tumour types (26,33,34), its precise function and role in medulloblastoma are not well understood. RASSF1A encodes a 340-amino acid protein that is predicted to contain a distal Ras-association domain, although its significance is not clear, and a recent study has suggested that RASSF1A plays a role in control of cell cycle arrest through cyclin D1 regulation (35). The RASSF1A gene is the only gene examined so far in this and other studies in medulloblastoma (which in total have examined the methylation status of 21 genes) (5,27), to be frequently fully methylated in a wholly tumour-specific manner, highlighting the importance of a better understanding of its function and role in the development of this tumour.

p14ARF and p16INK4a showed apparently tumour-specific partial methylation in a small number of tumours (2/44 and 3/44, respectively). p14ARF and p16INK4a are encoded by alternatively spliced RNA products from the same locus on chromosome 9p21. p14ARF regulates TP53 activity whereas p16INK4a is involved in cell cycle control (see ref. 36). Although the promoters of these two genes are in close proximity they show independent rather than concordant methylation. Promoter methylation of p14ARF and p16INK4a may contribute to tumour formation in a small subset of cases.

This study has also highlighted HIC1 and CASP8 as genes whose epigenetic silencing may have a potential role in the pathogenesis of medulloblastoma, as both genes show complete methylation in a significant proportion of tumours compared with partial methylation in the normal cerebellum.

HIC1 encodes a zinc finger transcription factor that is proposed to function as a transcriptional repressor (37). Recently, it has been shown that mice that are disrupted for one allele of HIC1 develop a range of malignant tumours (38), further supporting its putative role as a TSG. Our finding that HIC1 is partially methylated in normal cerebella, and that this methylation is increased in some tumours, corroborates by an alternative method the work of Rood (10), who examined HIC1 promoter methylation by NotI digestion and Southern blotting in normal brain and medulloblastoma samples. Importantly, we show here that complete methylation of the CpG sites examined in our study is correlated with a methylation-dependent decrease in expression of the HIC1 transcript in medulloblastoma cell lines. HIC1 maps to 17p13.3. Abnormalities of chromosome 17 are the most frequent observations in cytogenetic studies of medulloblastoma, and have been associated with a poor prognosis (39). Losses of 17p are observed in 30–50% of cases, and frequently encompass a common region of deletion at 17p13.3, suggesting the probable location of a medulloblastoma TSG at this locus (reviewed in ref. 2). The fact that HIC1 lies within this region and shows CpG island methylation associated with transcriptional silencing, together with its proven tumour suppressor and transcriptional repressor functions make it a strong candidate for the medulloblastoma TSG at 17p13.3. Further investigations are now required to clarify the relative contributions of normal tissue-specific methylation, de novo methylation and 17p deletion to HIC1 loss in medulloblastoma, and any functional role it may play in its pathogenesis.

Caspase 8 is an initiator caspase (cysteine-aspartyl-protease) involved in death receptor-mediated apoptosis and apoptosis triggered by other stimuli such as chemotherapeutic agents (reviewed in refs 40,41). Medulloblastoma cell lines and tumours show frequent methylation of its CpG island and methylation of the CpG sites examined is correlated with reduced or absent expression in medulloblastoma cell lines, suggesting that CASP8 methylation may play a role in abrogation of apoptotic pathways during medulloblastoma development. Complete methylation of CASP8 showed a statistically significant association with large cell and classic histopathological subtypes, but was not a feature of the nodular/desmoplastic subtype. These findings raise the possibility that methylation patterns in medulloblastoma may be predictive of clinicopathological features and of potential utility in patient management. The assessment of relationships between methylation events and patient prognosis was not appropriate in this retrospective study. Further studies in large prospective cohorts are now required to confirm these findings, and to fully establish any further clinical significance of all the tumour-specific methylation events identified in this study.

In summary, our results demonstrate that epigenetic inactivation of genes by promoter hypermethylation is a significant feature of medulloblastomas, and the frequent hypermethylation of RASSF1A, CASP8 and HIC1 relative to normal cerebella identifies these as gene- and tumour-specific events that may have an important role in medulloblastoma formation. The study of the epigenome has the potential to be a powerful tool in the identification of novel TSGs in medulloblastoma. Whilst a candidate gene approach has been important in demonstrating a role for epigenetic events in medulloblastoma, and in identifying a number of putative TSGs, which are potentially silenced in this tumour, the majority of genes investigated in such studies to date have not shown frequent tumour-specific methylation (this work, 5,27). Moreover such an approach is limited in the number of genes analysed and usually restricts analysis to genes that have shown methylation in other tumours. The employment of unbiased genome-wide techniques, such as CpG island microarrays (42), restriction landmark genome scanning (4) and the use of expression arrays to identify genes up regulated following demethylation treatments (43,44) may represent fruitful approaches for future identification of further novel TSGs epigenetically inactivated in medulloblastoma.


    Notes
 
* J.C.L. and M.E.L. contributed equally to this work. Back


    Acknowledgments
 
D384 Med, D425 Med and D458 Med were kind gifts from Dr D.Bigner, Duke University, Durham, North Carolina. MHH-MED-1 and MED-8A were kind gifts from Dr T.Pietsch, University of Bonn Medical Centre, Bonn, Germany. UW402 and UW228 were kind gifts from Dr J.Silber, University of Washington, Seattle. The remaining cell lines were obtained from the American Type Culture Collection. Normal cerebellar DNAs were a kind gift from Dr Michael Fruhwald, University of Muenster, Germany. Normal blood DNAs were obtained from the North Cumbria Community Genetics Project, UK. This work is supported by the North of England Children's Cancer Research Fund and The Katie Trust.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 6, 2003; revised November 6, 2003; accepted December 8, 2003.