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DNA methylation alterations of AXIN2 in serrated adenomas and colon
carcinomas with microsatellite instability
BMC Cancer 2014, 14:466doi:10.1186/1471-2407-14-466
Yuta Muto (firstname.lastname@example.org)
Takafumi Maeda (email@example.com)
Koichi Suzuki (firstname.lastname@example.org)
Takaharu Kato (email@example.com)
Fumiaki Watanabe (firstname.lastname@example.org)
Hidenori Kamiyama (email@example.com)
Masaaki Saito (firstname.lastname@example.org)
Kei Koizumi (email@example.com)
Yuichiro Miyaki (firstname.lastname@example.org)
Fumio Konishi (email@example.com)
Sergio Alonso (firstname.lastname@example.org)
Manuel Perucho (email@example.com)
Toshiki Rikiyama (firstname.lastname@example.org)
21 January 2014
16 June 2014
25 June 2014
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DNA methylation alterations of AXIN2 in serrated
adenomas and colon carcinomas with microsatellite
* Corresponding author
1 Department of Surgery, Saitama Medical Center, Jichi Medical University, 1-
847, Amanuma-cho, Omiya-ku, Saitama 330-8503, Japan
2 First Department of Surgery, Hamamatsu University School of Medicine, 1-20-
1, Handa-yama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan
3 Nerima-Hikarigaoka Hospital, 2-11-1, Hikarigaoka, Nerima-ku, Tokyo 179-
4 Sanford-Burnham Medical Research Institute (SBMRI), 10901 North Torrey
Pines Road, La Jolla, California, USA
5 Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Carretera
de Can Ruti S/N, 08916 Badalona, Barcelona, Spain
6 Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluis
Companys 23, Barcelona, Spain
* Corresponding author. Instituciò Catalana de Recerca i Estudis Avançats
(ICREA), Passeig Lluis Companys 23, Barcelona, Spain
† Equal contributors.
Recent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the
sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive
analyses of DNA methylation in SSA and MSI cancer have not been conducted.
With an array-based methylation sensitive amplified fragment length polymorphism (MS-
AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14
carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can
survey relative differences in methylation between normal and tumor tissues of 9,654 DNA
fragments containing all NotI sequences in the human genome.
Unsupervised clustering analysis of the genome-wide hypermethylation alterations revealed
no major differences between or within these groups of benign and malignant tumors
regardless of their location in intergenic, intragenic, promoter, or 3′end regions.
Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas.
Analysis of variance of DNA methylation between these four subgroups identified 56 probes
differentially altered. The hierarchical tree of this subset of probes revealed two distinct
clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and
Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation
(Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates
with APC and β-catenin in Wnt signaling, had more methylation alterations in Group 2, and
its expression levels negatively correlated with methylation determined by bisulfite
sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly
with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01).
Somatic methylation alterations of AXIN2, associated with changes in its expression, stratify
SSAs according to some clinico-pathological differences. We conclude that hypermethylation
of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation,
drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2
inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven
frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the
Colon sessile serrated adenoma, Microsatellite instability, BRAF mutation, DNA methylation,
Recent advances in colon cancer research have revealed a new pathological pathway distinct
from the traditional pathway, the tubular adenoma-carcinoma sequence . This alternative
pathway has been recognized as the serrated pathway, in which sessile serrated adenoma
(SSA) replaced the traditional tubular adenoma as the precursor lesion of a subset of
colorectal cancer .
SSA was identified as a new entity by Torlakovic et al. in 1996  and later classified in a
new category, the serrated polyps . The serrated polyps include hyperplastic polyps,
traditional serrated adenomas and sessile serrated adenomas, the characteristics of which are
serrated structure in the crypt epithelium [4-6]. Serrated polyp nomenclature is evolving and
interpretation of the literature is complicated by differing interpretations of the morphological
features of serrated polyps. Even among expert gastrointestinal pathologists there is
significant inter-observer variability in classification [7,8].
Regardless of the difficulty in the definition, recent research efforts led to recognize that
serrated polyps, especially SSA seemed to be precursor to many of the sporadic colorectal
cancers with microsatellite instability (MSI) . Mismatch repair deficiency leads to the
accumulation of hundred of thousands of somatic mutations in microsatellite sequences .
This mutator phenotype defined a specific molecular pathway for colon cancer because the
mutated cancer genes are in general different than those from cancers without MSI [10,11].
SSAs and MSI cancers were reported to exhibit similar features including predominant
location in the proximal colon, high BRAF and low KRAS mutation and enhanced DNA
Somatic hypermethylation of CpG islands in some genes includes the silencing of the MLH1
mutator gene and thus underlies many of the MSI sporadic cancers. Some investigators
conferred distinctive phenotypic and biological properties to the tumors displaying a so-
called CpG island methylator phenotype (CIMP), which was viewed as preceding the
development of a subset of MSI colon cancers [18,19]. However, no apparent bimodal
distribution was seen for the somatic hypermethylation alterations in gastrointestinal cancers
[20,21] thus challenging the CIMP hypothesis. Nearly 15 years later, the CIMP concept,
despite the publication of many CIMP papers (reviewed in ) still awaits for a clear
definition, including a stable set of CIMP markers, as well as for identification of the
underlying methylator gene(s) [22,23].
Despite of the elusive CIMP entity, the importance of somatic hypermethylation as
responsible for the silencing of several tumor suppressors and the MLH1 mutator gene, and as
a consequence the resulting MSI mutator phenotype, is highlighted by the evidence that SSA
display DNA methylation alterations that are frequently observed in MSI cancer [9,24-26].
However, comprehensive analyses of methylation alterations in SSA and MSI cancer have
not been conducted.
Methylation sensitive amplified fragment length polymorphism (MS-AFLP) is a
fingerprinting technique developed by Yamamoto et al. as a tool to analyze DNA methylation
in hundreds of loci simultaneously [27,28]. The approach utilized NotI restriction
endonuclease for targeting methylation changes in any of the two CpG sites within its
recognition sequence GCpGGCCpGC. Because nearly half of all NotI sites (44%) are located
in or adjacent to CpG islands, while the rest are located outside, MS-AFLP enabled to detect
both relative DNA hypermethylation and hypomethylation somatic alterations throughout the
genome. Comparing the intensity of the fingerprint bands from normal and tumor tissue DNA
provided an unbiased insight of the complex picture of those epigenetic alterations.
Employing this technique for the study of colorectal cancer we demonstrated that the MSI
phenotype was dominant over hypermethylation  and that some of the tumors without
MSI could be rationalized by an age-associated accumulation of DNA hypomethylation .
More recently, we developed a novel MS-AFLP array-based platform containing probes
consisting of 60-mer-oligonucleotides, which cover the sequences adjacent to all the 9645
NotI sites identified in the human genome . In this study, we performed a comprehensive
analysis of methylation alterations to characterize the epigenetic profiles of colon adenomas
and carcinomas of different genotype and phenotype to identify genes shared by these
Patients and tissues
Nineteen patients with sessile serrated adenoma (SSA), 8 with tubular adenoma (TA) and 26
with proximal colon cancer including 12 and 14 tumors with and without MSI, respectively
were recruited in this study. These were from the series analyzed in our previous study,
which had enough amount and high quality of DNA and RNA available for microarray
analysis . SSAs, TAs and colorectal cancer tissues were prospectively collected in Jichi
Medical University Hospital and Jichi Medical University Saitama Medical Center. SSA and
TA were obtained endoscopically and classified with two categories by the location, i.e.,
proximal and distal.
SSA was diagnosed by five architectural features; basal crypt serration, basal dilatation of the
crypts, crypts that run horizontal to the basement membrane, crypt branching and surface
villosity or papillarity as previously described [17,30-32]. When the endoscopically resected
polyp exhibited two or more features was diagnosed as SSA. Lesions showing typical
histological features of so-called “traditional serrated adenoma”  were excluded from the
Colon cancer tissues were obtained from patients who underwent surgical treatment. In all of
the lesions, a part of the tissue was taken in fresh and was frozen immediately for genetic
analysis and the rest of the tissue was used for histological analysis. Proximal lesions were
defined as proximal to splenic flexure, whereas distal lesions were defined as distal to splenic
flexure. All colorectal cancer tissues were collected from proximal colon. Written informed
consent for participation in the study was obtained from all participants. This study was
approved by Jichi Medical University Institutional Review Board.
DNA and RNA extraction
DNA was extracted by DNeasy® blood and tissue kit (Qiagen, Hilden, germany). Total RNA
was extracted from tissue culture cell lines by TRIzol® Plus RNA purification kit
(Invitrogen, Carlsbad, CA, USA).
BRAF and KRAS mutation analysis
BRAF (T1799A) and KRAS mutations were determined by direct sequencing after
polymerase chain reaction (PCR) amplification of exon 15 of the BRAF gene and codon 12
and 13 of the KRAS gene. For detection of the BRAF mutation, genomic DNA obtained from
fresh frozen samples was
GGCCAAAAATTTAATCAGTGGA-3′ primers. For the detection of the KRAS mutation, the
following primers were used: forward, 5′-CTGAAAATGACTGAATATAAACTTGT-3′ and
reverse, 5′-ATATGCATATTAAAACAAGATTTACC-3′ as described [17,33,34]. PCR
products were purified on a YM-30 Microcon column (Millipore) and sequenced using the
BigDye terminator v3.1 cycle sequencing kit on ABI Prism 3100 (both from Applied
amplified using: forward, 5′-
Genomic DNA was extracted from fresh frozen samples using the EZ1 DNA tissue kit
(Qiagen, Tokyo, Japan) and was amplified by PCR using the monomorphic markers BAT25
and BAT26 as previously described . PCR products were analyzed by Gene Scan using
ABI Prism 3100, and the sample was scored showing MSI if there were additional peaks in
the PCR products, or otherwise scored as microsatellite stable (MSS).
MLH1 and CpG island methylation
Combined bisulfite restriction analysis was performed to assess gene methylation using
primers that were designed to amplify the regions around the transcription start sites of the
target genes . Bisulfite modification was performed using the Epitect Bisulfite kit
(Qiagen), as described previously . Genomic DNA (1 µg) was used for conversion with
the bisulfite reagent. The primer sequences, annealing temperatures and restriction enzymes
utilized were identical to those previously described . After digestion, products were
electrophoresed on 2% agarose gels and stained with ethidium bromide. Methylation density
was confirmed using the image analysis program Image J, and positive methylation was
defined when the methylation- sensitive restriction enzyme digested ≥10% of the DNA .
Preparation, labeling and hybridization of DNA samples for MS-AFLP arrays
The genome-wide methylation profile was determined by a high-throughput array-based
analysis of methylation alterations. For this purpose, we introduced an array-based approach
of the methylation sensitive amplified fragment length polymorphism (MS-AFLP)
fingerprinting method, which can survey most of the 9654 DNA fragments containing all
NotI sequences in the genome, as previously described [27,38]. Genomic DNA was isolated
by QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The initial steps of the MS-AFLP
were performed as previously described [27,38]. Briefly, 1 µg of genomic DNA was digested
overnight with 5 units of methylation-sensitive NotI (Promega, Madison, WI, USA) and 2
units of methylation-insensitive Mse I (NE Biolabs, Beverly, MA, USA) at 37ºC. Two pairs
of oligonucleotides were annealed overnight at 37ºC to generate NotI (5′-
CTCGTAGACTGCGTAGG-3′ and 5′-GGCCCCTACGCAGTCTAC-3′) and Mse I (5′-
GACGATGAGTCCTGAG-3′ and 5′-TACTCAGGACTCAT-3′) specific adaptors.
The digested DNA was ligated in 1.25 µl each of 5 pmol/µl NotI and 50 pmol/µl Mse I
adaptor using 1 unit of T4 DNA ligase (Promega) overnight at 16ºC. The adaptor-ligated
template DNA was amplified by PCR using NotI (5′-GACTGCGTAGGGGCCGCG-3′) and
Mse I (5′-GATGAGTCCTGAGTAA-3′) primers. The PCR mixture consisted of 6 ng of NotI
primer, 30 ng of Mse I primer, 0.25 mM dNTP, and 1.5 unit of AmpliTaq DNA polymerase
(Applied Biosystems, Foster City, California, USA) in a final volume of 20 µl. The PCR
started at 72ºC for 30 s and 94ºC for 30 s, followed by 35 cycles of 94ºC for 30 s, 52ºC for 30
s, and 72ºC for 2 min. The final extension was performed for 10 min at 72ºC. The reactions
were then kept at 10ºC until the amplified DNA fragments were isolated using a QIA PCR
Clean-up kit (Qiagen). DNA was eluted into 50 µl of elution buffer.
Prior to hybridization on the MS-AFLP arrays, the DNA samples were differentially labeled
as previously described [27,38]. Briefly, fluorescently labeled fragments were prepared using
the Bioprime labeling system (Invitrogen). Each sample of PCR-amplified DNA (50 ng / 2.5
µl) was mixed with 5 µl of water and 5 µl of Random Primer Mix solution. The mixtures
were boiled at 100ºC for 2 min, quickly placed on ice for 1 min, and briefly centrifuged for
10 s. Then 1 µl of either CY5 Mix solution (1.56 mM each of dGTP, dATP and dTTP, 0.22
mM dCTP, and 0.11 mM Fluorolink CY5-dCTP) or CY3 Mix solution (1.56 mM each of
dGTP, dATP, and dTTP, 0.22 mM dCTP, and 0.11 mM Fluorolink CY3-dCTP) was added.
Fluorolink CY5-dCTP and CY3-dCTP were purchased from Amersham-Pharmacia. Klenow
fragment of E. coli DNA polymerase was then added to a final concentration of 0.8 U per µl.
The mixtures were incubated at 37ºC for 1 h before adding 2 µl of stop solution (0.5 M
EDTA) to terminate the reaction.
The CY5 and CY3 fluorescently labeled DNA fragments were separated from the
unincorporated dNTPs by filtration through Microcon YM-30 columns (Millipore, Bedford,
MA, USA). Each sample was reconstituted with 1× TE (pH 8.0) to a final volume of 37 µl,
and 2 µl of each sample was taken to determine the yield of labeled genomic DNA and the
specific activity after labeling and clean-up. Exposure of samples to light was minimized
during all experimental procedures.
The Cy3 and Cy5 labeled DNA samples were mixed in a siliconized tube with 70 µl of
Agilent 2× Hi-RPM Buffer (Agilent, Santa Clara, CA, USA). The mix was heated at 95ºC for
3 min and centrifuged at 6000 × g for 1 min to collect the sample at the bottom of the tube.
One hundred and ten µl of hybridization sample mixture was applied slowly to the gasket
slide into the Agilent SureHyb chamber base. Then, one microarray slide was placed onto the
gasket slide, with the active side facing down. The SureHyb chamber was covered onto the
slides, and the clamp assembly was slid onto both pieces. The assembled slide chamber was
placed in a rotator rack inside a hybridization oven and rotated at 20 rpm and hybridized at 65
°C for 40 hours. After hybridization, array slides were washed with Oligo aCGH Wash
Buffer 1 at room temperature for 5 minutes and Oligo aCGH Wash Buffer 2 at 37ºC for 1
min. To prevent Cy5 degradation by ozone, the slides were washed with acetonitrile for 30
seconds and then with Stabilization and Drying Solution for 30 seconds. The arrays were
scanned using an Agilent G2565BA DNA Microarray Scanner.
Quantitative reverse transcription-PCR
Tissue specimens were immediately added to RNA later (Ambion, Austin, TX, USA) and
stored at -80ºC until DNA or RNA extraction. Total RNA was immediately treated with
DNase I (Invitrogen, Carisbad, CA, USA) and reverse-transcribed using a Superscript II
reverse transcriptase kit (Invitrogen) to prepare first-strand cDNA. The primer sequences for
AXIN2 were 5′-CTGGCTCCAGAAGATCACAAAG-3′
ATCTCCTCAAACACCGCTCCA-3′ (reverse). Thermal cycling conditions were 42ºC for 60
min (cDNA synthesis), 95ºC for 30 sec (hot start), and then 40 cycles of 95ºC for 5 sec, 60ºC
for 30 sec, and 72ºC for 60 sec. The expression level of AXIN2 was determined using the
fluorescence intensity measurements from the ABI 7900HT Real-Time PCR System Data
Analysis Software. An ACTB fragment was amplified as an internal control.
(forward) and 5′-
Bisulfite sequencing analysis
DNA sequencing was performed after bisulfite modification, as previously described .
The primers for the bisulfite sequencing were 5′-TTGTATATAGTTTAGYGGTTGGG-3′
(forward) and 5′-AAATCTAAACTCCCTACACACTT -3′ (reverse). PCR was performed for
45 cycles, consisting of denaturation at 95ºC for 30 sec, annealing at 58ºC for 30 sec, and
extension at 72ºC for 60 sec, followed by a final 7-min extension at 72ºC for all primer sets.
The sequences were subjected to a BLAST search to determine their location in the genome.
Fisher’s exact was used to examine associations between two categorical variables.
Continuous variable comparisons between two groups were performed with the Student’s t-
test for those variables following a normal distribution, or with the non-parametric Mann–
Whitney-Wilcoxon test for those variables that do not follow a normal distribution. The level
of statistical significance was set at P < 0.05, unless otherwise specified. To determine the
significant genes from multiple samples, variance of analysis (ANOVA) with Bonferroni
correction was carried out using MeV , by which hierarchical clustering sample and gene
trees were also drawn, simultaneously. The threshold of significance was determined by
Bonferroni correction set at P = 0.05. To account for the bias due to the partial gene
representation in the MS-AFLP Array, all the gene enrichment analyses were performed
using the list of the genes present in the array as a background, instead of the total number of
genes in the human genome .
Clinicopathological and molecular features of samples
The clinicopathological and molecular features of the four subgroups of tumors, tubular
adenomas (TA) sessile serrated adenomas (SSA), and MSI and MSS carcinomas, are
summarized in Table 1. Patient gender and stage were not significantly different between
each group. Patients with MSI cancers were older than those with SSA (P = 0.01). MSI
associated with poorly differentiated phenotype. KRAS mutation was more prevalent in MSSs
and TAs as compared to MSIs and SSAs (P < 0.01), whereas BRAF mutation was
preferentially observed in MSIs and SSAs as compared to MSSs and TAs (P < 0.01). MLH1
methylation was detected only in MSI carcinomas.
Table 1 Clinicopathological and molecular data of colon adenomas and carcinomas
TA (n = 8)
SSA (n = 19)
65.4 ± 4.4 60.6 ± 9.8
Gender (M / F)
4 / 4 13 / 6
(A or B / C)
(W-M / P)
4 / 8 0 / 19
(mut / total)
0 / 8 19 / 19
(mut / total)
hMLH1 Methylation ( + / total )
0 / 8 0 / 19
1.8 ± 1.4% 1.2 ± 0.6%
0.6 ± 0.4% 0.3 ± 0.2%
1 Tumor grade. Well (W), moderately (M) or poorly (P) differentiated.
2 Hypermethylation and hypomethylation indicate the percentage of MS-AFLP array probes with values surpassing the
hypermethylation and hypomethylation thresholds, respectively.
3 For categorical data, p-values were calculated by χ2 test when comparing four groups, or by Fisher’s exact test when
comparing two groups. For continuous data, we applied one-way ANOVA followed by Tukey’s HSD multi-hypothesis
testing correction. The most Statistically significant p-value after correction corresponded always to the SSA vs MSI
comparison (a). In hypomethylation, a significant difference between SSA and MSS was also found (P = 0.0014). P-values
below 0.05 are in bold type.
NA: Not applicable.
MSS (n = 12)
62.2 ± 8.2
6 / 6
7 / 5
MSI (n = 13)
70.8 ± 11.3
6 / 7
11 / 2
12 / 0 9 / 4 0.096
4 / 12 0 / 13
4 / 12 9 / 13
0 / 12
1.2 ± 0.6%
1.0 ± 0.5%
5 / 13
1.9 ± 1.2%
1.0 ± 0.6%
Genome-wide surveillance of methylation alterations by MS-AFLP revealed no significant
differences between the 4 groups (Table 1). Also, there was no significant difference in the
overall frequency of hypermethylation alterations (including intragenic and intergenic
regions) and in the promoter and 3′end regions (Figure 1). There was a borderline difference
in methylation frequency at intragenic and 3′end regions (P = 0.035 and P = 0.041,
respectively) between SSAs and MSI carcinomas due to the higher number of alterations of
the later (Additional file 1: Figure S1). Regarding hypomethylation, SSAs displayed fewer
alterations than MSS and MSI carcinomas overall and in the different gene regions although
the differences were more pronounced compared with the MSI carcinomas (Figure 1 and
Additional file 1: Figure S1).
Figure 1 Frequency of hypermethylation (left) and hypomethylation (right) estimated
by MS-AFLP arrays. TA, in light blue: tubular adenomas. SSA, in dark blue: sessile
serrated adenomas. MSS, in yellow: microsatellite stable carcinomas. MSI, in orange:
microsatellite instable carcinomas. Frequencies were calculated as the percentage of probes
with log2 ratio value below −1.5 (for hypermethylation) or above 1.5 (for hypomethylation),
after filtering the 30% lower-intensity probes from each array. Top graphs, results including
all probes after filtering (13,515 probes per array). Bottom graphs, results considering only
the probes within ±2.5Kb of the 5′ end of genes (range: 7,924 to 8,035 probes per array). P-
values were calculated by one-way ANOVA followed by Tukey’s HSD multi-hypothesis
testing correction. Only p-values below 0.05 are shown.
Since the MS-AFLP array covers all NotI sites in the genome, these results extend our
previous findings with the MS-AFLP DNA fingerprinting lower resolution approach showing
that the original method reflected a panoramic view of the somatic methylation alterations
undergone by colon cancers at NotI sites.
Differentially methylated loci in TA, SSA, MSS and MSI
To identify the possible existence of distinct methylation profiles specific for each of the four
different tumor subgroups, unsupervised hierarchical clustering was performed using 9,645
probe sets, but the results revealed no clear differences (data not shown). We, then, carried
out an analysis of variance (ANOVA) to determine whether there were particular loci
specifically associated with these tumor subgroups, especially with serrated adenomas. This
analysis resulted in the identification of 56 distinctive probes, corresponding to 35 genes, 5
putative loci and 12 intergenic sequences (Figure 2 and Additional file 1: Table S1) that
appeared altered differentially among these subgroups.
Figure 2 Clustering of the samples according to their methylation profile.
Hypermethylation is indicated in red. Hypomethylation is indicated in blue. Samples are
shown on top of the heatmap. In white, the normal tissue DNA mix used as reference. Colors
for the four tumor groups are as in Figure 1. On the right side of the heatmap, the genes
associated to MS-AFLP probes. AXIN2 probes are indicated in black. Clustering was
performed by complete linkage using Pearson’s correlation on a subset of MS-AFLP probes
previously selected by ANOVA. Group 1 contains mostly tubular adenomas and MSS
carcinomas. Group 2A contains the majority of MSI carcinomas, and 5 MSS carcinomas. All
the sessile serrated adenomas are grouped in 2B1 and 2B2. Below the heatmap, in black,
cases positive for mutation in BRAF or KRAS, or hypermethylation of hMLH1.
The ANOVA-constructed hierarchical tree revealed two distinct subsets of samples (Groups
1 and 2, Figure 2). Seven MSSs (58.3%) and 2 MSIs (14.3%) cancers, as well as 7 TAs
(87.5%) were assigned to Group 1, while 12 MSIs (85.7%), all 19 SSAs (100%), 5 MSSs
(41.7%) and 1 TA (12.5%) to Group 2. Group 1 thus, consisted of many tumors participating
in the tubular adenoma-carcinoma pathway (TAs and MSSs cancers), whereas Group 2
included many SSAs and MSI cancers (Figure 2).
Distinct methylation profiles associates with SSAs including AXIN2
The clustering into two distinct groups by the ANOVA approach allowed performing t-test
analysis of these two groups (Additional file 1: Figure S2). The constructed hierarchical tree
revealed distinct epigenetic profiles, one of which was shared by many of the MSI cancers
and SSAs, and another, which was shared by MSS carcinomas and TAs. T-test also identified
168 probes that distinguished these tumors of the serrated-MSI cancer pathway and the
tubular adenoma-MSS carcinoma pathway (Additional file 1: Figure S2). AXIN2 was one of
these genes, which displayed a distinct level of methylation alterations between the two
groups (black bar at right margins of Figure 2 and Additional file 1: Figure S2).
Group 2 was further classified into two subgroups, one of which included most MSI cancers
(71.4%, Group-2A), and the other which contained all the SSAs (100%, Group-2B) (Figure
2). Group 1 displayed high frequency of KRAS mutation whereas Group 2 exhibited high
frequency of BRAF mutation (Figure 2 bottom). While Group 2 harbored many of MSI
cancers and all SSAs, as expected, methylation of MLH1 was only seen in MSI cancers in
Group 2A. Group 2B was also subdivided into two groups, 2B-1 and 2B-2, according to the
hierarchical tree (Figure 2 bottom).
Expression of AXIN2 associates with methylation alterations
ANOVA analysis identified AXIN2, which plays an important role in Wnt signaling pathway
cooperating with APC and b-catenin, being more frequently altered in Group 2 that included
the serrated adenomas. We measured the abundance of the corresponding AXIN2 mRNA in
the original SSAs and MSI cancers (Group 2) by quantitative reverse transcription–
polymerase chain reaction (RT–PCR). The expression levels were variable, with some tumors
showing little or no expression, while others exhibited a relatively high expression (Figure 3
Figure 3 Left: AXIN2 mRNA expression level of samples selected from cluster 2A, 2B1
and 2B2. Expression levels were analyzed by Q-PCR using ACTB housekeeping gene for
normalization. Thick horizontal lines within the boxes indicate the median value. Thin dashed
horizontal lines indicate the mean value. Middle: bisulfite sequencing results of the 0.5Kb
upstream region of the first exon of AXIN2. CpG sites are represented by a white or a black
circles for unmethylated or methylated sites, respectively. Right: AXIN2 mRNA expression
level in colorectal cancer cell lines HCT116 (MSI, white bar) and Caco2 (MSS, stripped bar),
and tumors, both sessile serrated adenomas (black bars) and MSI carcinomas (white bars),
ordered from lower to higher (top to bottom). Arrows between middle and right panel
connect those cases for which both the bisulfite sequencing and the mRNA expression results
are shown in the figure.
When the expression levels of AXIN2 were compared between the different groups according
to the hierarchical tree (Figure 2), the groups 2A and 2B-1 exhibited low expression levels of
AXIN2, whereas group 2B-2 had significant higher levels of expression of AXIN2 (Figure 3,
left). To examine whether the decreased levels of AXIN2 mRNA was linked to aberrant
methylation, the degree of methylation alterations of 12 CpG sites within the AXIN2 promoter
region (Figure 3 middle) was assessed in 5 plasmid clones of each of several samples from
groups 2A, 2B-1 and 2B-2. The AXIN2 promoter appeared more methylated in samples from
Group 2A than in Groups 2B-1 and 2B-2 (Figure 3 middle). Also, a tumor cell line with high
methylation (HCT116) exhibited lower expression of AXIN2 than another cell line (Caco2)
with little methylation (Figure 3, top of middle and right panels).
Expression of AXIN2 associates with different clinicopathological features of
The clinicopathological features of SSAs were analyzed in regards to the observed
differences in AXIN2 methylation and expression of the two subgroups 2B-1 and 2B-2. The
results are shown in Table 2. No differences were evident between these two groups in age,
gender, presence of carcinoma in the adenoma, or mucinous phenotype. However, the
adenomas with high methylation and low expression (Group 2B-1) were significantly
smaller, were more distal, and exhibited less crypt branching than those adenomas with low
levels of methylation and high levels of expression (Group 2B-2).
Table 2 Comparison of SSAs according to AXIN2 expression status
Group 2B-1 (n = 8)
Gender (Male / Female)
4 / 4
Patient Age (years)
62.8 ± 9.9
Tumor Size (mm)
9.4 ± 1.9
Location (C-A / T) 1
3 / 5
Crypt Branching 2
0 / 8
( + / total )
8 / 8
( + / total )
Carcinoma in adenoma
0 / 8
( + / total )
1 Anatomical location. Cecum (C). Ascending (A). Transversal (T).
2 Crypt branching was defined by the appearance of splits or fissures in the base of the crypts.
3 Hypermucinous appearance.
4 P-values were calculated by Fisher’s exact test for categorical variables and unpaired t-test
for continuous variables. P-values below 0.05 are in bold type.
Group 2B-2 (n = 11)
9 / 2
59.1 ± 9.9
15.3 ± 6.1
10 / 1
8 / 11
11 / 11 1.00
1 / 11 1.00
In this study, genome-wide surveillance of hypermethylation alterations in NotI sites by
MSFLP-array revealed that somatic hypomethylation was lower in SSAs compared with MSI
or MSS carcinomas. These benign tumors also occurred in younger individuals compared
with MSI carcinomas. This is consistent with the proposed hypothesis of demethylation as a
gradual accumulation of methylation replication errors during aging  assuming SSAs
being the precursors of the MSI carcinomas. In contrast, there were no major differences in
global hypermethylation between these groups of benign and malignant tumors regardless of
their location in intergenic, intragenic, promoter, or 3′end regions. Unsupervised clustering
analysis revealed no clear differences in the patterns of hypermethylatuon between or within
the four different tumor groups. Only after applying an ANOVA approach was possible to
discern that MSS cancers and TAs shared similar epigenetic features, so did MSI and SSA, as
reported previously [12-17]. The study also disclosed distinct profiles of genes relevant for
colorectal cancer such as homeobox genes, transcription factors, growth factors and genes in
the Wnt signaling pathway, including AXIN2.
Several papers estimated the frequency of Wnt signaling activation in SSAs but they are
controversial [40-44]. Possible explanations to account for the discrepancies may include that
some SSAs were misdiagnosed and wrongly categorized due to the complication in the
definition of serrated polyps . Therefore, a standardized diagnosis of SSA formulated
recently [30,32] was applied in this study.
Recent genome-scale exome sequencing analysis of 276 colorectal tumors, DNA copy
number, promoter methylation and messenger RNA and microRNA expression conducted by
the Cancer Genome Atlas project,  indicated that 92% of MSI cancer and 97% of MSS
cancers exhibited at least one alteration of genes involved in the Wnt pathway including
LRP5, FZD10, FAM123B, AXIN2, APC, CTNNB1 (β-catenin), TCF7L2, FBXW7 and SOX7.
Thus, Wnt signaling pathway seems to play a critical role in colorectal carcinogenesis in
general, although the spectrum of alterations may vary depending on the distinct oncogenic
AXIN was identified as a component of the complex in Wnt signaling pathway to regulate
the levels of β-catenin along with the wild type of adenomatous polyposis coli (APC) gene
. AXIN1 plays as a scaffold protein on which the complex for phosphorylation of β-
catenin by glycogen synthase kinase-3β (GSK-3β) is assembled . AXIN2 / Conductin was
identified as an AXIN homolog, which also played a scaffold protein, and was found mutated
in a subset of colorectal cancers [47,49]. AXIN1 appears to be a constitutive component of β-
catenin degradation complex for maintenance of basal life activity while AXIN2 is considered
to be an inducible component that is upregulated in response to increases in β-catenin levels
and thus serves to limit the duration and intensity of the Wnt signal [50,51]. AXIN2 has been
only found expressed in colon tissues (Additional file 1: Figure S3).
Epigenetic silencing of AXIN2 in MSI colon cancer was reported in 2006 . However,
aberrant methylation of AXIN2 in SSA has not been previously reported. In addition, we
identified an apparent increase in methylation alterations of AXIN2 from SSAs to MSI
carcinomas, suggesting that its expression deregulation by methylation associates with the
serrated adenoma-MSI cancer pathway.
The hierarchical tree identified three clusters according to methylation profiles, MSI, SSAs
epigenetically close to MSI and SSAs far from MSI. The expression levels of AXIN2 in these
three groups associated with the levels of methylation of AXIN2 in each group, respectively
(Figure 3). Our results suggest that expanding of methylation in the promoter region of
AXIN2 in SSAs lead to the suppression of the AXIN2 gene expression gradually, which
contributes to a stepwise acquisition of the epigenetic features seen in MSI colon cancer.
Koinuma et al.  reported that overexpression of AXIN2, either by treatment with 5′-
azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in a MSI CRC
cell line, which supports the functional significance of AXIN2 changes in methylation and
expression in our study. Dong et al.  reported progressive methylation of several genes
during the serrated pathway. In contrast with the epigenetic silencing of AXIN2 in MSI colon
cancer, up-regulation of AXIN2 mRNA was reported in MSS cancers. Indeed, in our study,
AXIN2 was frequently hypomethylated in MSS cancers, suggesting that the epigenetic change
of AXIN2 specifically associates with the MSI pathway for colon cancer. The fact that down-
regulation is not always accompanied by methylation (Figure 3) shows that additional
mechanisms may be at play to inactivate the suppressor function of the AXIN2 protein. For
instance, frameshift mutations of AXIN2 in MSI colon cancers may be one such additional
The epigenetic influence on MSI manifestation is shown by the hypermethylation and
silencing of MLH1 . High level of hypermethylation has been also associated with MSI
cancers [19,56], and also in SSAs [9,13,15,16,24-26,57,58]. However, MLH1 methylation
was not detectable in SSAs in contrast with the common presence observed in MSI cancers.
This shows that the epigenetic silencing of MLH1 is not involved in SSA development where
it must occur sometime during the adenoma expansion. But silencing of MLH1 then appears
to drive the adenoma cells towards the carcinoma state by the generation of many subsequent
mutations. The difference in age between the patients with SSAs and MSI carcinomas also
supports this suggestion, implying a necessary additional step after SSA development for the
accumulation of oncogenic mutations responsible for the carcinoma transition.
AXIN2 aberrant methylation appears to occur during adenoma growth like MLH1
methylation. The assumption here is that no methylation of MLH1 is found at the SSA stage
because once it occurs it may lead to the carcinoma transition in the absence of further clonal
expansion, since mutator genes do not alter the growth properties of the cells. The association
observed between aberrant methylation and down-regulation with small size SSAs without
crypt branching could be interpreted assuming that the occurrence of MLH1 methylation may
speed the transition to carcinoma in the absence of a need for further expansion of the
In conclusion, this study revealed that methylation aberrations likely play a role in the
serrated adenoma-MSI carcinoma sequence in colon cancer. Although the samples in this
study are too limited to draw definitive conclusions in some genetic or epigenetic
comparisons, other differences were sufficiently large to reach statistical significance. MLH1
silencing seem to occur in an already developed serrated adenoma by the previous occurrence
of somatic mutation in the BRAF oncogene. Once the serrated adenoma has evolved,
additional somatic alterations altering Wnt signaling, such as AXIN2 methylation or
frameshift mutation, may contribute to the adenoma’s further growth. Other genes besides
AXIN2, were identified that exhibit methylation profiles shared between SSA and MSI CRC
and would be interesting to further investigate how these genes work and interact with each
other during the progression of colon cancer of the serrated adenoma-MSI carcinoma
sequence. Nevertheless, when, contemporary with these somatic alterations, aberrant
methylation of the MLH1 gene occurs, this appear to be the determinant event in those cases
that eventually progress to the carcinoma stage by providing the cells with a strong mutator
Competing interests Download full-text
The authors declare that they have no competing interests.
Experimental design: Y.Muto, TM, KS, FK, MP, TR. Pathology analyses: TM. Molecular
analyses (i.e., MSI and methylation analyses, bisulfite sequencing, etc.) Y.Muto, TM, KS,
MS, TK, FW, HK. MS-AFLP Array design: KS, SA. MS-AFLP implementation, data
acquisition and analysis: TM, KS, HK, MS, KK, Y.Miyaki, SA, MP. Statistical analysis:
Y.Muto, KS, SA. Experimental work supervision and coordination: KS, FK, MP, TR.
Interpretation of the data: Y.Muto, TM, KS, TK, SA, MP, TR. Manuscript writing: Y.Muto,
TM, KS, TK, SA, MP. All authors read and approved the final manuscript.
This work was supported in part by a grant-in-aid for post graduate students from Jichi
Medical University, a grant-in-aid from the Ministry of Education, Culture, Sports, Science
and Technology, the JKA Foundation through its promotion funds from keirin Racing, and
Saitamaken Geka Ikai. MP and SA were supported by grants from the Spanish Institute of
Health Carlos III (FIS PI09/2444 and FIS PI12/00511).
1. Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 1990,
2. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP: Serrated polyps of the large intestine:
a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005,
3. Torlakovic E, Snover DC: Serrated adenomatous polyposis in humans.
Gastroenterology 1996, 110:748–755.
4. Longacre TA, Fenoglio-Preiser CM: Mixed hyperplastic adenomatous polyps/serrated
adenomas. A distinct form of colorectal neoplasia. Am J Surg Pathol 1990, 14:524–537.
5. Torlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM: Morphologic
reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003, 27:65–81.
6. Torlakovic EE, Gomez JD, Driman DK, Parfitt JR, Wang C, Benerjee T, Snover DC:
Sessile Serrated Adenoma (SSA) vs. Traditional Serrated Adenoma (TSA). Am J Surg
Pathol 2008, 32:21–29.
7. Farris AB, Misdraji J, Srivastava A, Muzikansky A, Deshpande V, Lauwers GY, Mino-
Kenudson M: Sessile Serrated Adenoma: Challenging Discrimination From Other
Serrated Colonic Polyps. Am J Surg Pathol 2008, 32:30–35.