Background
Colorectal cancer (CRC) is one of the most common cancers in the Western world and is marked by a high mortality rate [
1]. Early detection of CRC is the key to improved survival rates [
2]. Another factor affecting disease prognosis is CRC subtype [
3]. The microsatellite instability (MSI) subtype of CRC accounts for approximately 15 % of colorectal cancers [
4]. MSI tumors are distinguished by defects in the DNA mismatch repair system which leads to mutational insertions and deletions in short tandem repeats (microsatellites) of DNA [
5]. MSI is most often due to promoter hypermethylation and silencing of the
MutL homolog 1 (
MLH1) mismatch repair gene. Microsatellite stable (MSS) tumors account for 85 % of CRCs and exhibit chromosomal instability, including numerous chromosomal duplications, deletions and rearrangements [
6]. MSI tumors differ from MSS tumors in several ways; MSI CRCs exhibit proximal colonic location, increased lymphocytic infiltration, and poorer response to chemotherapeutic drugs [
7,
8]. MSI CRCs also demonstrate better prognosis at stages I-III, however, some studies suggest poor prognosis at stage IV, though metastatic MSI cases are rare [
7,
9]. A third CRC subtype, the CpG island methylator phenotype (CIMP), is characterized by widespread DNA hypermethylation of CpG-rich promoter islands. CIMP can exist concurrently with either the MSI or MSS phenotype, though it is more frequently found in tandem with MSI and
MLH1 hypermethylation [
10]. The prognostic significance of CIMP is currently undefined and may be modified by MSI status, presence of
BRAF mutation, tumor stage, or other factors [
11‐
13]. Recently, a classification system for further subtyping of CRC has been proposed, consisting of four subtypes [
14]. One subtype consists mostly of MSI cases, while the other three are able to categorize the remainder of cases by Wnt signaling activation, metabolic dysregulation, or mesenchymal activation.
The vast majority (up to 94 %) of CRCs feature dysregulation in the Wnt signaling pathway [
15]. Wnt signaling is important in normal development, cell growth and proliferation, but when inappropriately activated may also lead to tumor initiation and development [
16]. In canonical Wnt signaling, β-catenin accumulates within the cell, enters the nucleus and activates transcription of target genes, such as c-Myc and
ITF2 (immunoglobulin transcription factor 2) [
17,
18].
ITF2 is also known as
transcription factor 4 (
TCF4). In the absence of Wnt signaling, a β-catenin destruction complex including adenomatous polyposis coli (APC) targets β-catenin for ubiquitination followed by proteasomal degradation [
17,
18]. In many cases of CRC, the
APC gene is mutated, rendering it incapable of binding to β-catenin, which leads to β-catenin accumulation followed by its nuclear translocation and subsequent activation of downstream target genes [
18].
Evidence for DNA methylation of the
APC promoter has been found in CRC. However, to what extent
APC methylation plays a role in colorectal carcinogenesis is unclear, as a broad range of methylation levels has been found in the literature, from 11 up to 63 % of tumors methylated [
19‐
23]. Conflicting reports exist regarding the extent of
APC methylation in MSI CRCs. Some small-scale studies (MSI
n ≤ 29) have suggested that
APC methylation may be associated with the MSI subtype, but others show no significant difference [
21‐
27]. Still another study has found
APC methylation to be inversely correlated with CIMP but not MSI [
28].
The role of
ITF2, a Wnt signaling target gene, is less understood in CRC. It is a target of Wnt signaling and is overexpressed in colon cancers with Wnt dysregulation [
29]. Its expression was reported elevated in some cancers with aberrant Wnt signaling activation but decreased in others [
30,
31]. Among gastrointestinal malignancies,
ITF2 methylation has been reported in gastric cancer, but its methylation status has not been investigated in CRC [
32,
33].
Our group has previously demonstrated associations between the methylation status of key Wnt signaling pathway regulatory genes and CRC subtype including the extracellular Wnt antagonists
DKK1 and
SFRP1 as well as
Wnt5a which is involved in non-canonical Wnt activity [
34,
35].
In this study, we have examined the role of APC and ITF2 methylation in two nested case-case studies in CRC cohorts. These patients were recruited from two distinct Canadian populations and the case groups were stratified by their MSI status.
Discussion
Understanding the genetic and epigenetic differences amongst colorectal cancer subtypes is essential, as CRC subtypes differ in their treatment options and offer distinct survival outcomes. The Wnt signaling pathway is dysregulated in a majority of colorectal tumors and can be altered at the extracellular, intracellular and gene target level [
15,
34,
35]. We have shown that these changes to the Wnt pathway also differ based upon microsatellite instability status. We quantified the methylation status of the
ITF2 and
APC promoter CpG islands in a nested case-case study in two cohorts of colorectal carcinoma from two different populations, comparing cases by MSI status. We have demonstrated that the
ITF2 promoter is hypermethylated in tumor tissues compared with matched normal mucosa, and further, MSI-H tumors are more likely to incur promoter methylation compared with MSS tumors.
ITF2 promoter methylation was also significantly associated with
MLH1 promoter methylation, a common occurrence in MSI-H tumors. Conversely, we found that
APC, an important intracellular regulator of Wnt signaling marked by both genetic mutations and hypermethylation in CRC, acquires DNA methylation equally across subtypes.
This is the first study to investigate DNA methylation of
ITF2 in CRC cases. Here, we have established that
ITF2 methylation is a tumor-associated event, being a rare occurrence in normal tissue DNA. One sample out of 47 normal colonic tissue samples was methylated, but this rare occurrence may possibly be due to the field effect, or field cancerization, in which apparently normal cells acquire genetic and/or epigenetic alterations and may eventually progress to cancer. With regards to tumor methylation of
ITF2, we showed that it is associated with the MSI-H phenotype.
ITF2 has been reported to be a tumor suppressor that can induce cell cycle arrest and is sometimes lost due to loss of heterozygosity at 18q21 [
31]. However,
ITF2 expression has been found to be upregulated in some cancers with aberrantly activated Wnt signaling but decreased in others [
30,
31]. Further research is required to elucidate the role of
ITF2 in tumorigenesis. Treatment of gastric cancer cell lines with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza) restored mRNA expression in cell lines that had hypermethylation demonstrating methylation-dependent regulation of this gene [
33]. Thus, transcriptional silencing in CRC through methylation would likely lead to a decrease in its cellular expression levels potentially contributing to tumorigenesis.
APC promoter methylation is rarely observed in normal colonic tissue compared with CRC tumor tissue in our study population, which replicates the findings of a recent meta-analysis [
44]. However, contrary to
ITF2 methylation, we did not see an association with MSI-H CRC or any other clinical features.
APC expression is at least partially regulated by DNA methylation, as its expression increases in CRC cell lines after treatment with 5-aza [
45]. Several other studies have investigated the correlation between
APC methylation and MSI with varying results. Studies have shown wide variation in overall APC methylation, regardless of subtype, from as low as 18 % to as high as 63.4 % [
19,
20]. Our results show a more moderate level of 34–40 % of cases methylated. Findings in the literature for the correlation between
APC and MSI are even less clear, with methylation in MSI-H tumors ranging from 14.3–72.7 % [
21,
26]. However, these studies analyzed small numbers of patient samples, with a maximum of 29 MSI tumors used [
24]. Our study, on the other hand, employed a total of 432 samples, 216 of which were MSI-H. This sample size is many times larger than any other of its kind, giving more statistical power and certainty to our results.
There are no differences between level of methylation at different stages of CRC diagnosis for either
APC or
ITF2, indicating these may be early epigenetic events in tumorigenesis. Additionally,
APC methylation has been detected in colon adenoma, further evidence that it is an early event [
46]. Detection of
APC may be further exploited as a potential biomarker by detection in other biospecimens, as its methylation has been detected in both stool and plasma [
47,
48]. Further investigation of the presence of
ITF2 methylation in adenomas should be undertaken, as well as whether its methylation can be detected in stool or plasma. This research will indicate the potential of utilizing
ITF2 and
APC, perhaps in combination with other methylation markers, as non-invasive stool- or plasma-based methylation markers for CRC detection and/or subtype discrimination.
Data from colon and rectal tumors from The Cancer Genome Atlas (TCGA) shows that
APC mutation rates differ among the 224 tumors sequenced by exome sequencing. TCGA data described hypermutated tumors, which have a mutation rate of 12/10
6 and consist mostly of MSI-H tumors. The prevalence of
APC mutation in these hypermutated tumors is 51 % [
15]. Alternatively, non-hypermutated tumors, defined by a mutation rate <8.24/10
6 and consisting mostly of MSS tumors, incurred
APC mutations in 81 % of cases [
15]. This disparity in
APC mutation rates may be explained by DNA methylation to inactivate
APC leading to constitutive ligand-independent Wnt signaling. In this same data set
ITF2 is genetically altered in only 3 % of tumors, thus, methylation is likely to play a larger role in
ITF2 dysregulation in cancer [
49,
50].
While MSI-H tumors are a largely well-defined subtype of CRC, MSS tumors comprise the majority of cases and exhibit a wide variety of molecular characteristics. Thus, there is an emerging research focus to further classify molecular subtypes of CRC. Recently, four consensus molecular subtypes were defined. The first subtype consists mostly of MSI cases [
14]. The remaining three subtypes are defined by ‘canonical’ Wnt and MYC activation, metabolic dysregulation, or mesenchymal activation. Our results indicated that some MSS cases incur methylation of the Wnt genes studied, so perhaps these cases belong to the subtype characterized by Wnt activation. It would be interesting to see which sub-classification the MSS cases used in this study belong to, and how
ITF2 or
APC methylation profiles differ among the four subtypes.
MSI-H tumors often overlap with CIMP-positive status. Thus, the association we see between MSI-H and
ITF2 methylation may in fact be part of the widespread hypermethylation of CpG islands that characterizes CIMP tumors. CIMP status information is unavailable for some Ontario cases and all Newfoundland cases utilized in this study, thus we do not have a complete picture of CIMP for our cohort. From our available data we did see a trend between CIMP-positive status and
ITF2 methylation among MSS cases. However, there were only ten cases in this group. From our current findings as well as previous investigation into epigenetic regulation of Wnt signalling genes we have found that dysregulation through aberrant methylation is implicated in all subtypes of CRC, not solely in CIMP-positive cases. APC, which abrogates Wnt signaling intracellularly, is methylated in a proportion of CRCs, regardless of subtype while
ITF2, a downstream target of Wnt signaling, is methylated more often in MSI-H tumors. Our lab has previously found that
DKK1 and
SFRP1 promoter methylation, coding for two extracellular Wnt antagonists, segregate strongly with different CRC subtypes.
DKK1 methylation is associated with the MSI-H phenotype and other MSI-associated features, while
SFRP1 methylation is associated with MSS tumors [
34]. We also found that
Wnt5a methylation, which codes for an extracellular ligand of the non-canonical Wnt pathway, is associated with MSI-H [
35]. These results were found in the same cohort of Ontario and Newfoundland patients used in this study. These observations underscore the importance of both Wnt signaling and the role of DNA methylation in CRC.
One limitation of this study to bear in mind is that only a subset of available MSS cases was chosen for analysis by matching to MSI-H cases by age quartile, stage and sex. Individuals with MSI-H CRC are generally a younger age, more frequently female, have a lower tumor stage and are more frequently CIMP-positive than those individuals with MSS tumors. Thus, the MSS cases analyzed in this study do not wholly represent all MSS cases from our Ontario and Newfoundland populations. Additionally, we did not select MSS cases from the entire Ontario cohort, but only a subset available at the time this study was undertaken. The subset that we selected from did not differ in age, sex, stage or CIMP rates from the entire cohort.
The strengths of our study include large sample size, the inclusion of two independent well-characterized population-based cohorts and the choice of technology. The use of MethyLight technology is superior to methylation-specific PCR (MSP) and offers several advantages including a quantitative, high-throughput methylation-specific real-time PCR-based technique, which is amenable to using small quantities of DNA extracted from archival tissue specimens. MSP is a more qualitative and subjective method that has been used in many prior studies of APC methylation.
Acknowledgements
This work was undertaken at the Department of Laboratory Medicine and Pathobiology at the University of Toronto, Toronto, Ontario, Canada and was conducted at the Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. We sincerely thank the investigators, staff, and participants of the Colon Cancer Family Registry for their dedicated contributions leading to this work. We gratefully acknowledge The Jeremy Jass Memorial Pathology Bank for the tissue samples and pathology data used in this study. This work was supported by grant UM1 CA167551 from the National Cancer Institute and through cooperative agreements with the following CCFR centers: Ontario Registry for Studies of Familial Colorectal Cancer (U01/U24 CA074783) and Australasian Colorectal Cancer Family Registry (U01/U24 CA097735). This work was also supported by a Team Grant from the Canadian Institutes of Health Research (CTP-79845) awarded to BB, JAK, SG, RCG, and PSP by the NCI under Request For Applications (CA-95-011). The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the Colon Cancer Family Registry (CCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government or CFR. AJS was supported by the Interdisciplinary Health Research Team Program studentship funded by the Canadian Institutes of Health Research, the Lunenfeld-Tanenbaum Research Institute Studentship at Mount Sinai Hospital, and the University of Toronto Fellowship award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AJS carried out the methylation analysis, performed the statistical analysis, and drafted the manuscript. DD and ED participated in patient recruitment and coordination. DDB and JPY performed mutation analysis. DW performed CIMP analysis. PSP, RCG, SG, and JRM participated in study design and patient recruitment. JAK participated in the design of the study and statistical analysis and interpretation of data. BB conceived the study, contributed in analysis and interpretation of results, and drafted the manuscript. All authors read and approved the final manuscript.