A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines

During the last decade, epigenetic changes have been reported in many cancers and they are now recognized to be at least as common as genetic changes [1]. Aberrant methylation of cytosine located within the dinucleotide CpG is by far the best-categorized epigenetic change. The genome of the cancer cell demonstrates global hypomethylation [2, 3] as well as regional promoter hypermethylation of several tumor suppressor genes [4]. Hypermethylation of selected CpG sites within CpG islands in the promoter region of genes is associated with loss of gene expression and is observed in both physiological conditions, such as X chromosome inactivation [5], and neoplasia [6]. By inactivating various tumor suppressor genes, this epigenetic modification can affect many important cellular processes, such as the cell cycle (RB, p15 ^INK4b , p16 ^INK4a ), the TP53 pathway (p14 ^ARF ), the WNT signaling pathway (APC, E-cadherin), DNA repair (MGMT, hMLH1, BRCA1), apoptosis (DAPK), and the metastasizing process (E-cadherin, TIMP3) (reviewed in [1, 7, 8]).

Development of colorectal cancer through various morphological stages has been linked to several genetic and epigenetic changes. The majority of carcinomas have several chromosomal aberrations, a phenotype often referred to as chromosomal instability. Approximately 15% of the tumors are near diploid but exhibit microsatellite instability (MSI), seen as genome-wide short nucleotide insertions and deletions [9]. This phenotype is caused by a defect DNA mismatch repair system [9]. Subgroups of both types of colorectal carcinomas reveal aberrant methylation of tumor suppressor genes leading to lack of expression [10, 11].

Human cancer cell lines are important tools in cancer research. Their commercial availability and unrestrained growth make them well suited for in vitro studies. Although many of the known genetic aberrations in colon cancer cell lines have been comprehensively described [12], several of these cell lines have not been analyzed for methylation status of pathogenetically important target genes.

The frequencies of both methylation and gene mutation differ among various studies of cell lines and primary tumors. The genome characteristics, profiles of gene mutations, and methylation status are rarely reported in the same samples, let alone in large series. In the present report we address these potentially connected pathogenetic mechanisms by presenting methylation profiles of a set of genes in a series of MSI and microsatellite stable (MSS) colon cancer cell lines and primary colorectal carcinomas. The methylation profiles are compared with various known genetic and clinicopathological features of the same series.

Tumor cell lines are commonly used as experimental tools in cancer research, including studies designed to assess epigenetic changes. But whereas the genetic aberrations of colon cancer cell lines have been comprehensively described [12], the methylation profiles of potential target genes in the same or similar cell lines are often described only sparingly. A literature survey of the 20 colon cancer cell lines and their methylation status analyzed in this study showed that some cell lines and genes had been extensively studied, whereas others were left undescribed (Table 1). For half of the cell lines included in the present study, both methylated and unmethylated alleles have been found for one or more of the genes studied. As non-neoplastic cells are not found in cultured cancer cell lines, this can not be caused by the presence of normal cells, and although several biological and technical explanations may exist, allele specific methylation seems the most likely interpretation [23, 34]. In contrast, admixture of normal cells, tumor heterogeneity and/or monoallelic methylation may explain the coexistence of unmethylated and methylated bands in primary tumors.

It has been debated for some time whether cell lines are more frequently methylated than primary tumors [35]. Regarding overall CpG island hypermethylation, cancer cell lines have in general demonstrated an increased frequency of hypermethylation compared with primary tumors [15]. However, only a limited number of the genes analyzed have shown a statistically significant difference in methylation frequency [15]. Among several cancer types examined, colon cancer cell lines have been shown to resemble the most their respective primary tumor in this respect [36]. For the cell lines and primary tumors included in this study, the fraction of MSI and MSS samples was about the same and we saw no statistical difference in the overall number of methylated target genes in colon cancer cell lines versus colorectal primary tumors. Seemingly, large methylation percentage differences for individual genes were seen (Table 2) but they were statistically significant only for p16 ^INK4a methylation, independent of MSI stratification. Comparisons of in vitro tumor cells with primary tumors of each subtype (MSS and MSI) have also shown similar frequencies of TP53, KRAS and APC mutations [12] and ploidy stem line [37], which further supports the conclusion that the in vitro system is a suitable experimental tool that closely reflect the in vivo situation.

Previously reported variations in promoter hypermethylation frequencies of different tumor suppressor genes in colorectal cancer can be explained by various ratios of MSI versus MSS samples in the series analyzed, different methods for analyzing methylation, the inter-individual variation in scoring of methylated samples, incomplete bisulphite modification, tumor heterogeneity, and the fact that different parts of the gene promoter region in question have been analyzed. In the present study, we used primer sets known to only detect methylation in tumor cells, never in normal tissues from the same patients [24, 31, 38–42]. The promoter hypermethylation in these areas has also shown an impressive correlation with lack of protein expression, confirming that these are essential regions for gene expression [24, 31, 38–42]. The hMLH1 primers we designed amplify a region of the promoter, in which methylation invariably correlates with the lack of hMLH1 expression [18, 43, 44]. Methylation of this region has only been detected in tumor cells and not in normal mucosa [18, 43, 44].

As expected, the MSI primary tumors showed more methylation overall than did the MSS group. However, this was only significant for the hMLH1 and p14 ^ARF genes, whereas the four additional genes analyzed revealed similar methylation frequencies in the MSS and MSI groups. Promoter methylation of the hMLH1 gene was, not surprisingly, found only in tumors and cell lines with MSI, not in the MSS samples. The MSS tumors and cell lines per definition contain functional hMLH1 protein, and transcriptional silencing of hMLH1 by hypermethylation is known to be the main cause of MSI in sporadic CRC [26, 28, 45]. Also p14 ^ARF methylation may have a specific role in MSI tumors, since it seems to be most often inactivated in tumors with wild type TP53 (see below). However, the relatively high methylation frequencies of the remaining analyzed genes, and also their overall similar frequency in MSI and MSS samples, imply that they are important in colorectal carcinogenesis independently of tumor site and MSI status.

Inactivation of tumor suppressor genes by promoter hypermethylation has been recognized to be at least as common as gene disruption by mutation in tumorigenesis [1]. Indeed, most types of primary tumors harbor several genes inactivated in this way and some genes, like p16 ^INK4a , have been reported to be methylated consistently in most tumor types analyzed [46]. In colorectal carcinomas, the reported p16 ^INK4a methylation frequencies vary from 18% [47] to 50 % [48] with most of the observations centered around 36–40% [11, 27, 46, 49–51], i.e., slightly higher than our result. Both p16 ^INK4a and p14 ^ARF are more commonly methylated in tumors with MSI than in MSS [10, 11, 51–53], although we found that the methylation frequency of p14 ^ARF is higher than that for p16 ^INK4a in MSI colorectal carcinomas.

The DNA repair protein MGMT is able to remove promutagenic alkyl groups from O⁶-guanine by an irreversible transfer to an internal cysteine residue [54]. Left unrepaired, the alkylated O⁶-guanine has a tendency to base pair with thymine during replication, thereby introducing a G:C to A:T transition mutation in the DNA [55]. Inactivating promoter hypermethylation of the MGMT gene has previously been reported to be associated with G:C to A:T mutations in the tumor suppressor gene TP53 [56] and the proto-oncogene KRAS [57, 58]. Our data support this assumption for TP53 but seemingly not for KRAS, although no certain conclusions can be drawn from the limited number of samples with G:C to A:T mutations.

The p14^ARF protein interacts in vivo with the MDM2 protein, neutralizing MDM2's inhibition of TP53 [59]. Less hypermethylation of p14 ^ARF in tumors with mutated TP53 than in tumors with wild type TP53 has been reported previously [24]. Additionally, several reports have described an inverse relationship between MSI and TP53 mutation in colorectal carcinomas [60–62]. The frequent methylation we report for the p14 ^ARF gene in MSI tumors with few TP53 mutations is in agreement with a recent study [53] and supports the existence of this alternative pathway for TP53 inactivation.

Inactivation of the APC gene is frequent in colorectal and other gastrointestinal carcinomas, usually by truncating mutations [63, 64]. An alternative mechanism to inactivate the gene in colorectal tumors is by promoter methylation, and we report a frequency of APC methylation in the upper range of what has been seen in previous studies [51, 65, 66]. Somatic mutations in APC are common in colorectal cancer [67, 68] and, similar to what has been seen by others [12, 22, 69], almost half of the tumors displaying APC mutations in our study were also methylated. We have not looked at allele-specific mutation, but methylation and mutation in the same tumor might reflect one mutated allele and methylation of the other, in accordance with Knudson's two hit hypothesis. This has previously been demonstrated for APC in colorectal cancer samples by Esteller et. al [65]. APC has a central role in the WNT signaling pathway, which is suggested to play a part in colorectal carcinogenesis by its constitutive activation. Activation of this pathway results in increased transcription levels of genes like MYC and CCND1 (cyclin D1) further stimulating cell proliferation [63]. Among the 52 successfully analyzed primary tumors in this study, 35 had altered APC caused by methylation (n = 17) and/or gene mutation (n = 26). The E-cadherin gene was also methylated in 14/34 tumors with altered APC, presumably further stimulating WNT signaling [63]. Interestingly, APC methylation seemed to be more common in Dukes B stage tumors.

The present study confirms that methylation of hMLH1 in sporadic carcinomas is associated with proximal tumor location in the large bowel [14, 21, 45, 70], as above 90% of the primary tumors harboring a methylated hMLH1 promoter were taken from the right side of the colon. An association between sporadic proximal colon carcinomas and methylation has also been reported for p16 ^INK4a and p14 ^ARF [14, 21, 45]. Among our 53 primary tumors, we can only confirm this statistically for p14 ^ARF . However, p16 ^INK4a demonstrated the same tendency. Both hMLH1 and p14 ^ARF are strongly associated with MSI and MSI is in turn strongly associated with proximal tumor location [71, 72], hence, it is not unexpected that the methylation of both genes is associated with proximal location.

When it comes to gene methylation and its association with other clinicopathological features, contradictory results have been reported. Our observation that methylation of p14 ^ARF does not exclude p16 ^INK4a methylation, is in accordance with previous studies [21, 24]. Correlation of p16 ^INK4a or p14 ^ARF methylation with female gender and increased age has been described in some studies [14, 47] but not in others [11, 21, 24]. We found such an association between female gender and methylation of p14 ^ARF and hMLH1, but not of p16 ^INK4a . We also found a weak association between p16 ^INK4a methylation and increasing age. This potential age-specific methylation was not confirmed for any of the other genes studied. The gender-associated methylation of hMLH1 has previously been described [73, 74] and might explain the increased prevalence of colorectal tumors of the MSI type in the female patient group [74].

Like Toyota et. al [51], we found no statistically significant associations between tumors with widespread methylation and age, gender, or stage of the colorectal cancer.

The data presented here demonstrate that multiple genes are methylated in colorectal carcinomas. This underlines the important role epigenetic inactivation of tumor suppressor genes plays during the process of tumor development. Epigenetic changes in colon cancer cell lines are overall comparable with those of primary carcinomas of the large bowel, which make the cell lines relevant models for the in vivo situation. The methylation profile of specific genes, in particular hMLH1 and p14 ^ARF , has strong associations with genetic and clinicopathological features and might be related to biologically distinct subsets of colorectal tumors.