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Carcinogenesis Advance Access originally published online on November 28, 2007
Carcinogenesis 2008 29(2):434-439; doi:10.1093/carcin/bgm270
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Integrated analysis of chromosomal, microsatellite and epigenetic instability in colorectal cancer identifies specific associations between promoter methylation of pivotal tumour suppressor and DNA repair genes and specific chromosomal alterations

Sarah Derks, Cindy Postma1, Beatriz Carvalho1, Sandra M. van den Bosch, Peter T.M. Moerkerk, James G. Herman2, Matty P. Weijenberg3, Adriaan P. de Bruïne, Gerrit A. Meijer1 and Manon van Engeland*

Department of Pathology, GROW - School for Oncology and developmental biology, Maastricht University, Maastricht, 6200 MD, The Netherlands
1 Department of Pathology, VU University Medical Centre, Amsterdam, 1007 MB, The Netherlands
2 Department of Oncology, The Sidney Kimmel Comprehensive Cancer Centre at Johns Hopkins, Baltimore, MD 21231, USA
3 Department of Epidemiology, GROW-School for Oncology and developmental biology, Maastricht University, Maastricht, 6200 MD, The Netherlands

* To whom correspondence should be addressed. Tel: +31 43 3874622; Fax: +31 43 3876613; Email: m.vanengeland{at}path.unimaas.nl


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Colorectal cancer (CRC) is a complex and heterogeneous disease in which genomic instability and DNA promoter methylation play important roles. The aim of this study was to investigate the relationship between chromosomal instability (CIN), microsatellite instability (MSI) and promoter methylation of CRC-associated genes. Therefore, 71 CRCs were analysed for CIN and MSI by comparative genomic hybridization and the mononucleotide marker BAT-26, respectively. Promoter methylation of the tumour suppressor and DNA repair genes hMLH1, O6-MGMT, APC, p14ARF, p16INK4A, RASSF1A, GATA-4, GATA-5 and CHFR was analysed using methylation-specific polymerase chain reaction. These integrative analyses showed that in CIN+ CRCs, promoter methylation of GATA-4 and p16INK4A was inversely related to chromosomal loss at 15q11–q21 and gain at 20q13, respectively (P values: 3.8 x 10–2 and 4.5 x 10–2, respectively). Interestingly, promoter methylation of RASSF1A, GATA-4, GATA-5 and CHFR, as well as a high methylation index (MI), was positively related to chromosomal gain at 8q23-qter (P values: 1.5 x 10–2, 3.8 x 10–2, 3.9 x 10–2, 4.9 x 10–2 and 8.2 x 10–3, respectively). MSI was associated with BRAF mutation, promoter methylation of hMLH1, APC and p16INK4A and a high MI (total number of methylated genes) (P values: 2.4 x 10–2, 2.5 x 10–3, 1.8 x 10–2, 4.6 x 10–2 and 1.0 x 10–2, respectively). Therefore, we conclude that promoter methylation of pivotal tumour suppressor and DNA repair genes is associated with specific patterns of chromosomal changes in CRC, which are different from methylation patterns in MSI tumours.

Abbreviations: CAE, cancer-associated event; CGH, comparative genomic hybridization; CIMP, CpG island methylator phenotype; CIN, chromosomal instability; CRC, colorectal cancer; MI, methylation index; MMR, mismatch repair; MSI, microsatellite instability; PCR, polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Colorectal cancer (CRC) is a complex and heterogeneous disease in which numerous genetic and epigenetic alterations have been identified. Multiple molecular pathways are probably to underlie colorectal carcinogenesis in which genetic instability is thought to be a driving force (1–3).

Approximately 85% of all CRCs are characterized by chromosomal instability (CIN), which describes a condition of aneuploidy characterized by DNA copy number gains and losses (that may cause loss of heterozygosity), as well as structural chromosomal alterations (4). Despite extensive research, the mechanism driving the process of numerical and structural CIN in CRC is not known.

Previously, we have shown that CIN does not constitute random genetic noise, but occurs in patterns of associated chromosomal changes (5,6). Seven specific chromosomal changes, losses in 8p21-pter, 15q11–q21, 17p12–13 and 18q12–21 and gains in 8q23-qter, 13q14–31 and 20q13 are strongly associated with the progression of colorectal adenomas towards carcinomas and can be considered as cancer-associated events (CAEs) (5).

In addition, ~10–15% of sporadic CRCs, and all tumours from patients with hereditary non-polyposis CRC, are characterized by microsatellite instability (MSI) (7). This form of genetic instability is associated with defective mismatch repair (MMR), which is commonly achieved by promoter methylation of the DNA MMR gene hMLH1 in sporadic CRCs (8–11), whereas in patients with hereditary non-polyposis CRC germline mutations of MMR genes take place (7). Defective MMR leads to accumulating frameshift mutations at simple repeated nucleotide sequences. Some of these mutations affect genes that are implicated in colorectal carcinogenesis such as TGFβRII, IGFIIR and BAX, thereby leading to a mutator phenotype (12–15).

Emerging evidence indicates that also ‘epigenetic instability’ is involved in CRC development (16–18). Besides altered chromatin structure and histone modifications, epigenetic instability is characterized by hypermethylation of multiple CpG islands, a phenotype also referred to as the CpG island methylator phenotype (CIMP).

Although the concept of CIMP is under debate (19) and different definitions of CIMP are used (20–23), emerging evidence supports the existence of a group of CRCs with a high prevalence of methylated CpG islands (17,20,24). Even though the relative importance of this pathway in tumour progression is not well understood, CIMP-positive tumours are thought to represent a distinct group of carcinomas that show a strong association with MSI and BRAF mutations (21,25) and an inverse relationship to CIN (26,27).

We showed previously that DNA promoter methylation of tumour suppressor and DNA repair genes hMLH1, O6-MGMT, APC, p14ARF, p16INK4A, RASSF1A, GATA-4, GATA-5 and CHFR is a frequent and early event in CRC (28). In this study, a high frequency of promoter methylation was already present in adenomas without any histological signs of progression, whereas morphologically normal mucosa from patients with carcinomas showed significantly less promoter methylation.

However, it is not directly clear how these methylation events are related to the different genetic phenotypes in CRC. Therefore, in the present study we investigated the relationship between promoter methylation of established tumour suppressor and DNA repair genes and CIN and MSI in a group of 71 CRCs. Data were complemented with analysis of BRAF mutation status.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Patient material
This study included 71 CRCs of which a subset has been analysed for chromosomal abnormalities by comparative genomic hybridization (CGH) previously (5). The CRC tissues were obtained from 67 patients, 34 males and 33 females (mean age: 67 years, range: 44–89 years). Archival material was used in compliance with the institution's ethical regulations. Four patients had synchronous carcinomas. Since this can influence results, all analyses were performed omitting these CRCs as well. Since no difference in significant results was observed, only results of the total series of 71 CRCs are reported. The clinical pathologic characteristics are listed in Table I. CRCs were categorized as proximal or distal tumours based on their location in relation to the splenic flexure. This series includes colon carcinomas and rectal carcinomas but no significant difference between distal colon carcinomas and rectal carcinomas regarding promoter methylation, chromosomal alterations and MSI was observed. Therefore, distal colon carcinomas and rectal carcinomas are reported as one group.


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  		Table I. Clinicopathologic characteristics of CIN and MSI carcinomas

 
CGH analysis
Fifty-one CRC tissue samples have been analysed by conventional CGH previously (5). Chromosomal gains or losses were interpreted when the fluorescence ratio was significantly higher or lower than 1.0, as evaluated by the 95% confidence interval.

Twenty additionally collected CRCs were analysed by array CGH analysis using 5 K bacterial artificial chromosome arrays (28–30). Briefly, we used a full-genome in-house printed array containing~5000 clones with an average resolution of 1 Mb. Protocols used for labelling, hybridization and feature extraction are available on http://internet-extra.vumc.nl/microarrays/. After applying a smoothing algorithm to the array CGH data (31), DNA copy number ratios obtained by array CGH were recoded as gains and losses at the resolution of whole chromosome arms compatible with the data obtained by chromosome CGH.

Since the presence of two or more specific chromosomal alterations (losses at 8p, 15q, 17p and 18q and gains at 8q, 13q and 20q) is strongly associated with CRC progression (5), we defined CIN by the presence of two or more of these CAEs.

MSI analysis
The MSI status of each tumour was evaluated by amplification of BAT-26, a single poly(A) tract shown previously to be highly sensitive and specific for MSI (32,33). A subset of CRCs (n = 46) was also analysed for MSI by amplifying four additional mononucleotide repeats in a pentaplex reaction as described previously (34). This pentaplex includes the mononucleotide repeats BAT-25, BAT-26, NR-21, NR-24 and NR-27. Primer sequences were as described previously (34). Each sense primer was end-labelled with one of the fluorescent markers FAM, HEX or NED. Pentaplex polymerase chain reaction (PCR) was performed with an initial 5 min denaturation step at 94°C, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 7 min. Amplified PCR products were run on an Applied Biosystems PRISM 3100 automated capillary electrophoresis DNA sequencer. Allelic sizes were estimated using Genescan 2.1 software (Applied Biosystems, Foster City, LA). The accordance between the BAT-26 analysis and pentaplex data was 100%. For the complete study population, MSI was therefore defined as instability of the BAT-26 locus.

Promoter methylation analysis
DNA methylation in the CpG islands of hMLH1, O6-MGMT, APC, p16INK4A, p14ARF, RASSF1A, GATA-4, GATA-5 and CHFR gene promoters was determined by chemical modification of genomic DNA with sodium bisulfite and subsequent methylation-specific PCR as described in detail elsewhere (28,35,36). Primer sequences are as described previously and available upon request (37–39). Unsupervised hierarchical cluster analysis (see also Data analyses section) was performed to cluster CRCs based on the methylation status of the above-mentioned genes and to study patterns of promoter methylation.

Detection of BRAF codon 599 mutations
The common BRAF V600E mutation in exon 15 was analysed by a semi-nested PCR and subsequent restriction fragment length polymorphism analysis as described previously (40–42). The semi-nested amplification step (primer sequences and PCR conditions are available upon request) was added in order to optimize the amplification of formalin-fixed, paraffin-embedded DNA.

Data analyses
Differences in frequencies, e.g. of gene methylation between different groups of carcinomas, were evaluated by the Pearson's {chi}2 or Fisher's exact test where appropriate. The Mann–Whitney U non-parametric and Kruskal–Wallis tests were used for comparing means of the continuous variables between groups of CRCs. We calculated means of the total number of methylated genes per case, referred to as methylation index (MI), which is defined as the number of genes methylated divided by the number of genes analysed.

All reported P values are two-sided, and a P value <0.05 was considered statistically significant, using SPSS software version 12.0. All statistical tests were corrected for multiple comparisons using the Bonferroni method.

In order to group CRCs based on the patterns of promoter methylation, we performed unsupervised hierarchical cluster analysis using Spotfire® Decisionsite 8.2 (Spotfire AB, Göteborg, Sweden). Data on methylation status of hMLH1, O6-MGMT, APC, p16INK4A, p14ARF, RASSF1A, GATA-4, GATA-5 and CHFR per tumour were entered and grouped with the setting ‘Ward's method’ for clustering, ‘half square Euclidean distance’ as similarity measure and row interpolation to handle missing values.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
CIN and MSI
We first categorized all CRCs based on their type of genomic instability, i.e. CIN and MSI.

We showed previously that the acquisition of two or more CAEs, defined as losses in 8p21-pter, 15q11–q21, 17p12–13 and 18q12–21 and gains in 8q23-qter, 13q14–31, and 20q13, is strongly associated with CRC progression (5). Therefore, we defined CIN+ as the presence of two or more CAEs, which were present in 55 of 71 CRCs (77.5%). In this CIN+ group, losses in 8p, 15q, 17p and 18q and gains in 8q, 13q and 20q were present in 54.5, 49.1, 67.5, 61.8, 54.5, 49.1 and 43.6% of CRCs, respectively (Table II).


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  		Table II. Epigenetic and genetic events in CIN and MSI carcinomas

 
MSI status, defined by BAT-26 instability, was assessable in 67 of 71 CRCs (94%). Eleven of the 67 CRCs (16.4%) were instable at the BAT-26 locus and classified as microsatellite instable (MSI+), whereas 56 of 67 CRCs (83.6%) displayed microsatellite stability (MSI–).

Although MSI+ CRCs displayed some chromosomal alterations (mean 5.7), a higher number of chromosomal alterations was observed in MSI– CRCs (mean: 12.2, P value: 2.7 x 10–3). When we examined associations between specific chromosomal alterations and microsatellite status, MSI– was associated with chromosomal losses in 1p, 15q, 17p and 18q and gain in 13q (P values: 1.8 x 10–2, 4.3 x 10–2, 1.3 x 10–2, 1.7 x 10–3 and 3.6 x 10–3, respectively). Four of these five chromosomal alterations belong to the group of CAEs, which were significantly more frequently present in MSI– CRCs (3.5 CAEs) when compared with MSI+ CRCs (0.9 CAE; P value, 5.0 x 10–5). Although not statistically significant, also CAEs loss of 8p and gain of 8q and 20q also occur more frequently in MSI– CRCs when compared with MSI+ CRCs.

In order to investigate whether an overlap exists between CIN and MSI CRCs, we correlated CIN status to MSI status. We classified the 67 CRCs, for which both CIN and MSI status could be determined, into four groups: CIN+/MSI– group (n = 50), CIN+/MSI+ group (n = 2), CIN–/MSI– group (n = 6) and CIN–/MSI+ group (n = 9). In this way, 88% of CRCs could be characterized by either CIN or MSI, and only in 3% (n = 2) of the CRCs, an overlap between the CIN and the MSI pathway was present.

Promoter methylation analysis
We next included gene promoter methylation into the analysis. As shown previously (28), 71 CRCs were analysed for promoter methylation of hMLH1, O6-MGMT, RASSF1A, APC, p16INK4A, p14ARF, GATA-4, GATA-5 and CHFR. We selected these genes since they are reported to be functionally and frequently methylated in CRC carcinogenesis. Promoter methylation was present in 46.5, 57.7, 26.8, 39.4, 40.8, 33.6, 80.3, 79.9 and 40.8% of CRCs (Table II), respectively.

Furthermore, relations between promoter methylation events were frequently observed for p16INK4A, GATA-4, GATA-5 and CHFR. Promoter methylation of p14ARF was associated with methylation of all genes analysed, except from hMLH1 and APC. Promoter methylation of hMLH1 was solely associated with CHFR promoter methylation (P value: 2.7 x 10–2). The same accounts for APC promoter methylation, which only showed an inverse relation to O6-MGMT promoter methylation (P value: 2.4 x 10–3) (data not shown).

Except from the association between hMLH1 and CHFR methylation, all relationships were independent of MSI or CIN status and were also observed when we analysed interrelations in CIN+ CRCs separately.

Similar patterns of promoter methylation were observed when we performed an unsupervised hierarchical cluster analysis, based on promoter methylation of the above-mentioned genes. Cluster analysis identified three groups of CRCs (Figure 1). We calculated the mean MI, number of genes methylated divided by the number of genes analysed per case, of each cluster. Cluster I (n = 15, 21.1%) showed the highest frequencies of promoter methylation (MI: 0.82), and clusters II and III could be characterized by a significantly lower MI index of 0.53 (n = 31, 43.7%) and 0.31 (n = 25, 35.2%), respectively (P value: 5.7 x 10–10). Although the majority of the genes show the highest frequency of promoter methylation in cluster I, O6-MGMT is most frequently methylated in cluster II (P value: 7.1 x 10–6) and APC is frequently methylated in cluster I as well as in cluster III, whereas only 3.2% of CRCs in cluster II show APC promoter methylation (P value: 3.77 x 10–7) (Table III).


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  		Table III. Epigenetic and genetic events in clusters of methylated genes

 


Figure 1
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  		Fig. 1. Unsupervised hierarchical clustering based on promoter methylation of nine DNA repair and tumour suppressor genes. Cluster analysis resulted in the identification of 3 groups of CRCs with different patterns of methylated genes. A black box represents a methylated gene promoter, a white box represents an unmethylated promoter and a grey box illustrates a failed PCR amplification.

 
CIN and MSI in relation to promoter methylation
We next studied how promoter methylation was related to CIN and MSI.

We determined the mean MI of the four subgroups of CRCs defined above and observed that the CIN–/MSI+ and CIN+/MSI+ groups of CRCs showed the highest MI (both 0.72), whereas CIN–/MSI– and CIN+/MSI– CRCs showed a MI of 0.58 and 0.46, respectively (P value: 4.0 x 10–2) (Figure 2).


Figure 2
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  		Fig. 2. Promoter methylation in distinct CRC groups. Promoter methylation has been analysed for nine DNA repair and tumour suppressor genes. Mean MI, total number of methylated genes divided by the number of genes analysed per group; CIN–, chromosomal stable; CIN+, chromosomal instable; MSI–, microsatellite stable and MSI+, microsatellite instable. *P value = 0.04.

 
In addition to the relation between MSI and a high mean MI, MSI+ was associated with promoter methylation of hMLH1 (P value: 2.5 x 10–3), APC (P value: 1.8 x 10–2) and p16INK4A (P value: 4.6 x 10–2) (Table II). Although not statistically significant, promoter methylation of RASSF1A, p14ARF, GATA-5 and CHFR occurred more frequently in MSI+ CRCs. GATA-4 was methylated in 100% of the MSI+ cases versus 76.4% of the MSI– cases. O6-MGMT methylation on the other hand did not differ between MSI+ and MSI– CRCs.

CIN+ CRCs displayed a significantly lower MI when compared with CIN– CRCs (MI: 0.47 and 0.66, respectively; P value: 6.3 x 10–3) and an inverse relation to promoter methylation of hMLH1, p16INK4A and CHFR (P values: 4.2 x 10–2, 3.1 x 10–2 and 3.0 x 10–2, respectively). Furthermore, p16INK4A methylation showed an inverse relationship to CAEs chromosomal loss of 18q and gain of 20q and the total number of CAEs (P value: 5.7 x 10–3, 1.9 x 10–2 and 3.9 x 10–2, respectively). In addition, GATA-4 methylation shows an inverse relation to CAE chromosomal loss of 15q and, although not statistically significant, to the total number of chromosomal abnormalities (P value: 1.2 x 10–2 and 5.9 x 10–2, respectively) (data not shown).

Although MSI+ CRCs show a higher MI when compared with CIN+ CRCs, promoter methylation events are present in CIN+ CRCs. Therefore, we studied the relationship between gene promoter methylation events and chromosomal alterations in CIN+/MSI– CRCs (n = 50). Also, in this group the inverse relationship between p16INK4A and gain of 20q (P value: 4.5 x 10–2) and GATA-4 methylation and loss of 15q (P value: 3.8 x 10–2) was observed. Furthermore, a positive relationship between promoter methylation of RASSF1A, GATA-4, GATA-5 and CHFR and CAE gain of 8q was present (P values: 1.5 x 10–2, 3.8 x 10–2, 3.9 x 10–2 and 4.9 x 10–2, respectively). Although not statistically significant, the same accounts for promoter methylation of hMLH1, O6-MGMT, APC, p16INK4A and p14ARF. Additionally, CRCs with gain of 8q show a higher MI (0.55) when compared with CRCs without 8q gain (MI: 0.38; P value: 8.2 x 10–3). No other associations between CAEs and MI were observed.

BRAF mutation shows an inverse relation to CIN
BRAF mutation status was determined in 57 of 71 CRCs, of which sufficient DNA were available. Seven CRCs (12.3%) exhibited the V599E BRAF mutation.

We investigated the association between BRAF mutation and CIN and observed an inverse relation approaching statistical significance (P value: 5.4 x 10–2) (Table IV). In addition, BRAF-mutated CRCs display significantly less CAE when compared with wild-type BRAF CRCs (1.6 CAE and 3.3 CAE, respectively; P value: 2.9 x 10–2). Furthermore, BRAF mutation was associated with MSI+ since 36.4% of the MSI cases and only 6.8% of the MSI– cases showed a BRAF mutation (P value: 2.4 x 10–2). All BRAF-mutated CRCs showed hMLH1 promoter methylation as well as an association with promoter methylation of p14ARF and CHFR (P values: 1.1 x 10–2, 3.6 x 10–2 and 2.7 x 10–2, respectively).


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  		Table IV. CIN, MSI and promoter methylation in relation to BRAF mutation

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The aim of the present study was to investigate the association between CIN, MSI and DNA promoter methylation of multiple DNA repair and tumour suppressor genes reported to be involved and frequently methylated in CRC carcinogenesis.

In order to perform such a study, clear definitions for the genetic phenotypes are needed. MSI was defined by instability of the BAT-26 locus, as was shown previously to be highly sensitive and specific for MSI-H (32,33). Defining CIN was less obvious since no generally accepted definition of CIN exists, despite the common use of the term CIN in the literature. Biologically, CIN is a dynamic process and is usually defined as an accelerated rate of chromosome missegregation during cell division (3). Since no common definition of CIN is present, we defined CIN, in this study, as the presence of two or more CAEs. CAEs are defined as chromosomal alterations, losses in 8p21-pter, 15q11–q21, 17p12–13 and 18q12–21 and gains in 8q23-qter, 13q14–31 and 20q13, which show a strong association with progression of adenomas towards carcinomas (5). Using this definition, CIN was observed in 77.5% of CRCs, a percentage which is in line with several other reports using other definitions (1,3,43).

When we studied the association between chromosomal alterations and MSI, MSI showed an inverse relation to chromosomal losses in 1p, 15q, 17p and 18q and gain in 13q, of which four were CAEs. Chromosomal loss in 1p has been shown previously to be strongly associated with aneuploidy and also the inverse relationships between MSI and chromosomal losses in 15q, 17p and 18q and gain in 13q are in agreement with the earlier reports (44–47). Although chromosomal aberrations are not completely absent in MSI+ CRCs (45,46), with the definition of CIN used in the present study, the overlap with MSI was only 3%.

In this study, we investigated promoter methylation of genes relevant to pathogenesis of CRC, i.e. promoter methylation of hMLH1, O6-MGMT, APC, p14ARF, p16INK4A, RASSF1A, GATA-4, GATA-5 and CHFR and observed some interesting findings. Although most of the genes showed concordant methylation, APC promoter methylation showed an inverse relation to O6-MGMT methylation and hMLH1 was solely associated with CHFR promoter methylation. Thereby, we confirmed the association between hMLH1 and CHFR methylation that have been shown previously in both CRC and gastric cancer (39,48,49).

We performed a hierarchical cluster analysis to study the patterns of promoter methylated genes and observed that not all genes show the highest frequency of promoter methylation in the group with the highest number of methylated genes. O6-MGMT was most frequently methylated in a group of CRCs displaying moderate frequencies of methylated genes, whereas APC promoter methylation was most frequently observed in the groups of CRCs with the highest and lowest frequencies of methylated genes. These results are in agreement with the recent reports that show that O6-MGMT promoter methylation is associated with CIMP-low CRCs (50) and that APC promoter methylation shows an inverse relation with features of CIMP (51). Together, these results indicate that promoter methylation of specific genes might occur in different pathways.

When we studied the association between promoter methylation and genetic alterations, MSI was associated with hMLH1, p16INK4A and APC promoter methylation. The association between promoter methylation of hMLH1 and MSI, as well as the associations with BRAF mutation, is well described (8–11) and also the association of p16INK4A methylation and MSI has been reported previously (20). However, the relationship between APC methylation and MSI is not directly clear. APC is a key regulator of the Wnt signalling pathway and downregulation of APC is one of the earliest events observed in colorectal carcinogenesis that leads to activation of Wnt signalling. Inactivation of APC is accomplished by promoter methylation, loss of heterozygosity or mutation. Although genetic and epigenetic events can collaborate for inactivation of this gene (52), we and others have previously observed that APC mutation and APC promoter methylation occur in distinct CRCs (28,53). This is in line with the observation of Samowitz et al. (54), which showed an association between APC mutations and microsatellite stable tumours, whereas this study shows a relationship between APC promoter methylation and MSI CRCs.

Furthermore, a recent study has shown an inverse relationship between CIMP, defined by promoter methylation of MINTs, (Methylated in Tumours), p14ARF, p16INK4A and hMLH1, and CIN analysed by loss of heterozygosity analysis (26). We also observed lower frequencies of methylated genes in CIN CRCs when compared with MSI CRCs and an inverse relationship between promoter methylation of p16INK4A and CAEs chromosomal loss at 18q12–21 and gain at 20q13, as well as the total number of CAE was observed. Moreover, GATA-4 promoter methylation showed an inverse relation to CAE chromosomal loss at 15q11–q21, which indicates that promoter methylation and CIN occur in distinct tumours.

However, we also observed a statistical significant positive relationship between CAE chromosomal gain of 8q23-qter and promoter methylation of RASSF1A, GATA-4, GATA-5 and CHFR. Although not statistically significant, the same accounts for promoter methylation of hMLH1, O6-MGMT, APC, p16INK4A and p14ARF. Amplification of this chromosomal region involves the proto-oncogene MYC that encodes a transcription factor playing a critical role in regulating cell growth, proliferation, apoptosis and differentiation through its ability to activate or repress transcription (55). The finding that myc represses transcription through recruitment of DNA methyltransferase corepressor that leads to methylation and silencing of target genes (56) might provide a mechanistical link between amplification of 8q23-qter and an increased number of methylated genes in CRC.

In summary, our results indicate that CIN and MSI CRCs can be considered as two alternative mechanisms. Although promoter methylation shows a strong relationship to MSI, promoter methylation of pivotal tumour suppressor and DNA repair genes is observed in CIN CRCs and is associated with a specific pattern of chromosomal changes.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Transnational University Limburg and the Dutch Cancer Society (KWFVU02-2618).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received August 13, 2007; revised October 12, 2007; accepted November 17, 2007.


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