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Carcinogenesis. May 2011; 32(5): 741–747.
Published online Feb 8, 2011. doi:  10.1093/carcin/bgr020
PMCID: PMC3140140
Promoter methylation of Wnt antagonists DKK1 and SFRP1 is associated with opposing tumor subtypes in two large populations of colorectal cancer patients
James B. Rawson,1,2 Michael Manno,3 Miralem Mrkonjic,1,2 Darshana Daftary,3 Elizabeth Dicks,4 Daniel D. Buchanan,5 H. Banfield Younghusband,4 Patrick S. Parfrey,4 Joanne P. Young,5 Aaron Pollett,8 Roger C. Green,4 Steven Gallinger,1,2,3,6 John R. McLaughlin,2,3,7 Julia A. Knight,2,7 and Bharati Bapat1,2,8*
1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A1, Canada
2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, L6-304B, Box 30, Toronto, Ontario M5G 1X5, Canada
3Ontario Familial Colorectal Cancer Registry, Cancer Care Ontario, Toronto, Ontario M5G 2L7, Canada
4Faculty of Medicine, Memorial University, St. John's, Newfoundland A1C 5S7, Canada
5Familial Cancer Laboratory, Queensland Institute of Medical Research, Herston, Australia QLD 4006
6Department of Surgery, University of Toronto, Ontario M5S 1A1, Canada
7Dalla Lana School of Public Health, University of Toronto, Ontario M5S 2S1, Canada
8Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
*To whom correspondence should be addressed. Tel: +1 416 586 4800 ext. 5175; Fax: +1 416 361 2655; Email: bapat/at/lunenfeld.ca.
Received November 1, 2010; Revised January 12, 2011; Accepted January 29, 2011.
Aberrant activation of canonical Wnt signaling is a hallmark event in colorectal carcinogenesis. The Dickkopf-1 (DKK1) and Secreted Frizzled Related Protein 1 (SFRP1) genes encode extracellular inhibitors of Wnt signaling that are frequently silenced by promoter hypermethylation in colorectal cancer (CRC). These methylation events have been identified as prognostic markers of patient outcome and tumor subtype in several cancers but similar roles in CRC have not been comprehensively examined. In CRC, the microsatellite instability (MSI) subtype associates with favorable disease outcome but the molecular events that are responsible remain poorly understood. Consequently, we quantified promoter methylation status of the Wnt antagonist genes DKK1 and SFRP1 in a large population-based cohort of CRCs from Ontario (n = 549) and Newfoundland (n = 696) stratified by MSI status. We examined the association between methylation status and clinicopathlogical features including tumor MSI status and patient survival. DKK1 and SFRP1 were methylated in 13 and 95% of CRCs, respectively. In Ontario, DKK1 methylation was strongly associated with MSI tumors after adjustment for age, sex and tumor location [odds ratio (OR) = 13.7, 95% confidence interval (CI) = 7.8–24.2, P < 0.001]. Conversely, SFRP1 methylation was inversely associated with MSI tumors after these adjustments (OR = 0.3, 95% CI = 0.1–0.9, P = 0.009). Similar results were obtained in Newfoundland. There were no independent associations with recurrence-free survival. This is the first large study to identify associations between Wnt antagonist promoter hypermethylation and CRC MSI subtype. These events provide insight into subtype-specific epigenetic mediation of Wnt signaling in CRC.
Colorectal cancer (CRC) is a heterogeneous disease that is influenced by a wide range of genetic and epigenetic events. As the second leading cause of cancer mortality in North America [1,2], substantial effort has been directed toward classifying these molecular events into distinct tumors subtypes with the goal of identifying new therapeutic strategies and more robust predictors of patient outcome. CRC tumors are currently classified into clinically relevant subtypes by DNA microsatellite instability (MSI) status and, more recently, by CpG island methylator phenotype (CIMP) status. MSI tumors are caused by dysfunctional DNA mismatch repair and are found in 10–20% of CRCs (3). These tumors exhibit favorable prognosis compared with their microsatellite stable (MSS) counterparts despite poor response to 5-fluorouracil-based chemotherapy (4,5). CIMP tumors are characterized by widespread DNA hypermethylation of CpG-rich promoter islands and are found in 15–20% of sporadic CRCs (6). CIMP maintains conflicting roles in prediction and prognostication of CRC. Although epigenetic alterations are prevalent in both of these subtypes, the specific epigenetic events that drive subtype-specific outcome remain poorly understood and there remains significant interest in identifying events that may have prognostic, predictive or therapeutic potential for CRC.
Perhaps the most important pathway in CRC is Wnt signaling. Wnt signaling consists of the canonical/β-catenin pathway as well as several non-canonical/β-catenin-independent pathways including planar cell polarity and Wnt/Ca2+. The canonical pathway has a well-established role in colorectal oncogenesis, driving proliferation and dedifferentiation in 90% of CRCs (7). The function and status of non-canonical Wnt pathways, however, are less well characterized in CRC, having been implicated in both tumor suppression and oncogenesis (reviewed in refs 8,9). Important emerging mediators of both types of Wnt signaling are the secreted Wnt antagonists: Secreted Frizzled Related Proteins (SFRP1-5), Dickkopf (DKK1-4) and Wnt Inhibitory Factor-1 (WIF-1). These glycoproteins have established tumor suppressive roles in CRC cell lines (10,11) and xenografts (12) but are often silenced by promoter CpG island hypermethylation in numerous solid (1317) and non-solid (18,19) tumors. Critically, these Wnt antagonist promoter methylation events have been recognized as strong prognostic markers of poor outcome in primary cancers of the kidney (20), blood (21), breast (22,23) and lung (24,25) but not yet in CRC where they are particularly common and seem to occur early during tumor formation (10,11,26). Intriguingly, both the frequency and prognostic significance of Wnt antagonist methylation can vary with tumor subtype in acute myeloid leukemia and non-small cell lung cancer (21,24); however, no large prognostic studies have examined the relationship between Wnt antagonist methylation and tumor subtype in CRC. Understanding these relationships may provide insight into tumor subtype-specific changes in Wnt pathways and may uncover associations between Wnt antagonist methylation and patient prognosis in CRC.
Consequently, we investigated promoter methylation levels of Wnt antagonist genes DKK1 and SFRP1 in a large cohort of MSI-stratified CRCs from two distinct Canadian populations: Ontario, a heterogenous population with moderate incidence of CRC, and Newfoundland, a founder population with high incidence of CRC but relatively low incidence of germ line mutations causing MSI (27). DKK1 methylation has been associated with CRC progression (12). SFRP1 is the most frequently methylated family member in CRC (11) and has been identified as a marker of high grade, late stage and metastases in renal cell carcinoma (26,28). We examined associations between methylation of these two genes and patient clinicopathological features with a focus on MSI subtype and outcome.
Study participants
Participants in this population-based study were cases of primary colorectal carcinoma recruited through the Ontario Familial Colorectal Cancer Registry (OFCCR) and Newfoundland Familial Colorectal Cancer Registry (NFCCR). Patient accrual, data collection and biospecimen collection procedures for OFCCR have been described previously in detail (29). Briefly, residents of Ontario diagnosed with pathology-confirmed primary CRC between the ages of 20 and 74 from 1997 to 2000 were eligible for recruitment. Patients were asked to complete several self-administered questionnaires and consent to accession of cancer-related medical records from which clinical data were abstracted including family history (30). Apparent cases of familial adenomatous polyposis were excluded. One thousand and four probands with blood and/or tissue biospecimens were recruited. Due to the high prevalence of self-reported Caucasians (92.5%), we excluded all non-white patients as well as those with unknown or mixed ethnic background (n = 83). We were able to obtain 600 tumor samples for methylation analysis, representing 561 of these probands. Twenty-two probands were subsequently excluded during methylation analysis due to poor quality DNA (see Materials and Methods: MethyLight), leading to a final cohort of 549 Ontario cases. Annual follow-up was available for these probands including vital status and recurrence status. All patient data were obtained through protocols approved by the research ethics boards of Mt Sinai Hospital and the University of Toronto.
The population-based case selection strategy followed by the NFCCR was similar to that of the OFCCR except for a slightly later recruitment period, 1999–2003 (31), and accrual of deceased patients by proxy consent from living family members. Seven hundred and forty seven probands were recruited by the NFCCR of which 721 probands were white (northern European origin) and had tumor samples available for methylation analysis. Methylation analysis was successfully performed on 696 cases after removal of poor quality samples. An overview of patient clinicopathological features is shown in Table I.
Table I.
Table I.
Distribution of clinicopathological features including Wnt antagonist methylation in primary colorectal carcinomas from Ontario and Newfoundland
Molecular analysis
DNA was extracted from archival paraffin-embedded tumors microdissected to enrich for tumor cells (>70% neoplastic cellularity) and used to assess MSI status, MLH1 methylation and BRAF V600E mutation. MSI testing was performed using National Cancer Institute guidelines, as described previously (30) using five or more markers: ACTC, BAT-25, BAT-26, BAT-40, BAT-34C4, D10S197, D18S55, D2S123, D5S346 and MYC-L. MSI status was defined by the number of positive markers: MSI high (≥30% unstable markers), MSI low (1–29% unstable markers) or MSS (0% unstable markers). In Newfoundland, tumors initially characterized as MSI low were screened for a second set of markers: BAT-40, D7S519, D17S787, D18S58 and D20S100 (27). Due to the limited frequency of MSI low in our populations, MSI low and MSS tumors were combined into one ground. MSI High tumors were subsequently denoted as ‘MSI’.
MLH1 methylation status was assessed by quantitative MethyLight assay as described previously (6). Positive methylation was defined as percent methylated reference >10.
In Ontario, the somatic T>A mutation at nucleotide 1799 causing the V600E mutation in the BRAF gene was determined using fluorescent allele-specific polymerase chain reaction (PCR) (32). Twenty to 50 ng of tumor DNA was amplified in a 25 μl reaction using 100 nM of allele-specific primers tagged with different fluorophores: (mutant forward) 6FAM-5′-CAGTGATTTTGGTCTAGCTTCAGA-3′; (wild-type forward) NED-5′-TGATTTTGGTCTAGCTACAGT-3′ and (common reverse primer) 5′-CTCAATTCTTACCATCCACAAAATG-3′). The mutant allele (A1799) PCR product was 97 bp, 3 bp larger than the wild-type allele (T1799) PCR product. After amplification, 1 μl of the PCR product was added to a 8.7 μl mixture of HiDi formamide and ROX Genescan 500 size marker (Applied Biosystems, Foster City, CA) for separation by capillary electrophoresis using an ABI 3100 genetic analyzer. Allelic discrimination was performed using GeneMarker v1.75 software (SoftGenetics) based on product size and fluorescence. Positive and negative controls were run in each experiment. Ten percent of samples were validated with 100% concordance. In Newfoundland, BRAF V600E mutational testing was performed by allele-specific PCR as described previously (33). Positive samples were validated by direct sequencing.
Immunohistochemical staining of mismatch repair proteins MLH1, MSH2, MSH6 and PMS2 was performed as described previously (34). Staining was classified as present, absent or inconclusive. Tumors with absent staining in any of these proteins were defined as mismatch repair deficient.
MethyLight
Methylation analysis was performed on 50 ng sample DNA by semiquantitative MethyLight assay (35). DNA was pretreated with sodium bisulphite using the EZ DNA Methylation Gold Kit (Zymo Research Corp, Orange, CA) according to the manufacturer’s protocol and stored at −20°C. Primers and probes were designed to amplify a region within the promoter CpG island of DKK1 and SFRP1 proximal to regions previously studied (12,28). Amplification of Alu-C4 was used to normalize for DNA input (36). Primer and probe sequences were as follows: (DKK1 forward) 5′-TTTGGGATCGTAGGGGGTTTTC-3′; (DKK1 reverse) 5′-AACCTAAATCCCCACGAAACCG-3′; (DKK1 probe) 6FAM-5′-TGATTTTGTAGTCGAATCGGT-3′-MGBNFQ; (SFRP1 forward) 5′-GAATTCGTTCGCGAGGGA-3′; (SFRP1 reverse) 5′-AAACGAACCGCACTCGTTACC-3′; (SFRP1 probe) 6FAM-5′-CCGTCACCGACGCGAAAACCAAT-3′-MGBNFQ; (ALU-C4 forward) 5′-GGTTAGGTATAGTGGTTTATATTTGTAATTTTAGTA-3′; (ALU-C4 reverse) 5′-ATTAACTAAACTAATCTTAAACTCCTAACCTCA-3′ and (ALU-C4 probe) 6FAM-5′-CCTACCTTAACCTCCC-3′-MGBNFQ.
Samples were analyzed in 96-well plates on an ABI 7500 RT-PCR thermocycler as described previously (36). Each gene was also amplified from exogenously methylated CpGenome lymphocyte DNA (Millipore, Billerica, MA). Methylation was calculated as percent methylated reference as follows (6): [gene/Alu-C4]sample/[gene/Alu-C4]CpGenome × 100%. Positive methylation was defined as percent methylated reference ≥10, a conservative value for transcriptional silencing in other genes (37). Due to the high frequency of positive SFRP1 methylation, an additional percent methylated reference cutoff value was defined at the third quartile for each population (ON = 74.43, NFLD = 55.33) to stratify samples into low and high methylation groups.
Validation was performed on randomly selected samples at monthly intervals. Reassignment of methylation status was exceptionally rare. During screening and validation, samples with an Alu-C4 threshold cycle >22 were considered poor quality, retreated with sodium bisulfite and reanalyzed to ensure robust amplification. Samples that remained poor quality were removed from the analysis (ON: n = 37; NFLD: n = 24).
Statistical analysis
Comparison of methylation status in matched tumor-normal pairs was assessed using McNemar's chi-squared test. Clinicopathological associations were examined by Fisher’s exact test. Multivariate modeling of MSI status was performed using unconditional logistic regression adjusted for age, sex and tumor location. Unadjusted associations of methylation to recurrence-free survival were assessed using Kaplan–Meier survival plots with the long-rank test. Multivariate recurrence-free survival models were performed using Cox regression adjusted for age, sex, stage and grade, with and without MSI status. Time to event was calculated from the date of diagnosis to the date of first recurrence/death or censored at the date of last contact. Two-sided P <0.05 was considered statistically significant. Bonferroni corrections for multiple comparisons was incorporated where appropriate. Analyses were performed in SPSS version 16.0 (SPSS Inc.).
Methylation analysis
We quantified methylation levels within the promoter regions of DKK1 and SFRP1 in 549 CRCs from Ontario and 696 CRCs from Newfoundland. In Ontario, methylation of DKK1 and SFRP1 was observed in 71 (13.0%) and 521 (94.9%) tumors, respectively (Table I). Similar frequencies were observed in Newfoundland, where DKK1 and SFRP1 were methylated in 94 (13.5%) and 655 (94.1%) tumors. These frequencies are comparable with those in other populations as reported by methyl-specific PCR (1012). Due to the high frequency of positive SFRP1 methylation and to take advantage of quantitative methylation analysis, SFRP1 methylation status was further classified into low and high methylation groups (see Materials and Methods: MethyLight). High methylation was observed in 141 (25.7%) tumors in Ontario and 174 (25.0%) tumors in Newfoundland.
To verify that these methylation events were specific to tumor tissue, we also quantified DKK1 and SFRP1 methylation levels in 100 normal colonic mucosa samples in parallel with their matched tumors. To increase the number of informative samples, oversampling was conducted for cases with positive tumor DKK1 methylation. Thirty-six cases had DKK1 methylation in tumor only, whereas no cases had methylation in normal only (McNemar's P < 10−10). Two cases were concordant for methylation in both tumor and normal tissue and the remaining 62 cases had no methylation in either tissue. Sixty-six cases had SFRP1 methylation in tumor only, whereas one case had methylation only in normal tissue (McNemar's P < 10−18). Thirty cases had both samples methylated and three had neither.
Methylation and clinicopathological features
We then examined associations between methylation status of DKK1 and SFRP1 patient clinicopathological features were examined in both populations. In Ontario, DKK1 methylation was much more common in MSI tumors than in MSS tumors [odds ratio (OR) = 13.7, 95% confidence interval (CI) = 7.8–24.2, P < 10−19] (Table II). This relationship was also reflected in associations between DKK1 methylation and several MSI-associated tumor characteristics, including the following: immunohistochemical deficiency of at least one mismatch repair (MMR) protein (‘MMR protein status’; OR = 15.7, 95% CI = 8.7–28.2, P < 10−19), MLH1 methylation (OR = 38.0, 95% CI = 18.7–77.1, P < 10−26), proximal location (OR = 4.3, 95% CI = 2.5–7.5, P < 10−7), mucinous histological type (OR = 2.4, 95% CI = 1.3–4.6, P = 0.01) and female gender (OR = 3.2, 95% CI = 1.9–5.6, P < 10−4) (Table II). There was no association between DKK1 methylation and inherited germ line mutations in mismatch repair genes that cause Lynch syndrome MSI tumors (‘MMR germ line mutation’ OR = 0.6, 95% CI = 0.1–2.6, P = 0.74). The absence of a relationship to Lynch syndrome-associated MSI tumors was maintained after adjustment for age (data not shown), indicating that there was no confounding effect of age-associated methylation. Indeed, DKK1 methylation was not associated with age ≥50 years compared with age <50 years (OR = 1.4, 95% CI = 0.6–3.4, P = .55). DKK1 methylation was also strongly associated with BRAF V600E mutation (OR = 48.7, 95% CI = 26.1–90.8, P < 10−41), a common alteration in MSI and CIMP. DKK1 methylation remained associated with BRAF V600E after adjustment for MSI status (OR = 22.4, 95% CI = 11.5–40.1, P < 10−21).
Table II.
Table II.
Associations between Wnt antagonist methylation and age, sex, MSI status, MSI-associated features and BRAF V600E status in Ontario
In contrast, methylation status of SFRP1 exhibited clinicopathological associations opposite to those of DKK1. SFRP1 methylation was less common in MSI tumors compared with MSS tumors (OR = 0.3, 95% CI = 0.1–0.9, P = 1.33 × 10−4) and was negatively associated with MSI-associated characteristics: MMR protein deficiency (OR = 0.3, 95% CI = 0.1–0.9, P = 0.002), proximal location (OR = 0.5, 95% CI = 0.2–1.2, P = 0.05), female gender (OR = 0.3, 95% CI = 0.1–0.8, P = 0.02) and germ line MMR germ line mutation (OR = 0.2, 95% CI = 0.04–0.7, P = 0.008) (Table II). Compared with tumors with highly methylated SFRP1, tumors with low SFRP1 methylation tended to show less extreme odds ratios for these characteristics (supplementary Table 1 is available at Carcinogenesis Online). SFRP1 methylation was more common in cases with age ≥50 years compared with age <50 years (OR = 7.0, 95% CI = 2.5–19.7, P = 0.001), validating previous studies that have identified SFRP1 methylation as an age-associated epigenetic event (38,39). SFRP1 methylation was also associated with BRAF V600E (OR = 1.8, 95% CI = 0.2–15.1, P = 0.02).
A similar analysis was conducted in our validation population from Newfoundland. Again, DKK1 methylation was strongly associated with tumor MSI (OR = 9.4, 95% CI = 5.4–16.2, P < 10−14) and with the same set of MSI-associated characteristics: MMR protein deficiency (OR = 11.6, 95% CI = 6.6–20.6, P < 10−15), MLH1 methylation (OR = 93.0, 95% CI = 20.6–419.2, P < 10−14), proximal location (OR = 5.7, 95% CI = 3.4–9.4, P < 10−12), mucinous histological type (OR = 2.4, 95% CI = 1.4–4.1, P = 0.003) and female gender (OR = 2.1, 95% CI = 1.4–3.3, P < 0.001) but not germ line MMR gene mutation (OR = 0.9, 95% CI = 0.2–3.8, P = 1.0) or age >50 years (OR = 1.3, 95% CI = 0.6–2.7, P = 0.61). Again, DKK1 methylation was associated with BRAF V600E (OR = 48.7, 95% CI = 26.1–90.8, P < 10−41) and this association remained after adjustment for MSI status (OR = 12.2, 95% CI = 6.2–24.1, P < 10−12).
In Newfoundland, trends across SFRP1 methylation groups again showed an inverse association with MSI (OR = 0.2, 95% CI = 0.1–0.7, P = 0.009) and several MSI-associated characteristics: MMR protein deficiency (OR = 0.3, 95% CI = 0.1–0.8, P = 0.03), proximal location (OR = 0.4, 95% CI = 0.2–0.7, P = 0.009) and mucinous histological type (OR = 0.3, 95% CI = 0.1–0.7, P = 0.009) (Table III). SFRP1 methylation was not associated with age over versus under 50 years (OR = 2.8, 95% CI = 1.1–6.8, P = 0.08). Once more, tumors with low methylation tended to show less extreme odds ratios for these characteristics relative to highly methylated tumors (supplementary Table 2 is available at Carcinogenesis Online).
Table III.
Table III.
Associations between Wnt antagonist methylation and age, sex, MSI status, MSI-associated features and BRAF V600E status in Newfoundland
Methylation status of DKK1 or SFRP1 showed weak or no associations with several other clinicopathological characteristics in Newfoundland (supplementary Table 3 is available at Carcinogenesis Online) and Ontario (supplementary Table 4 is available at Carcinogenesis Online), including tumor stage, pT/pN/pM substage, grade, local invasion, patient history of irritable bowel syndrome and patient history of inflammatory bowel disease.
Methylation and multivariate MSI
We constructed a multivariate MSI model to adjust for the possible confounders of age, sex and tumor location (Table IV). These covariates were each individually associated with MSI status in both populations (data not shown). In Ontario, DKK1 methylation remained strongly associated with MSI in this model (OR = 10.7, 95% CI = 5.6–20.7, P < 10−11), whereas SFRP1 methylation remained inversely associated with MSI (OR = 0.3, 95% CI = 0.1–1.2, P = 0.009). In Newfoundland, DKK1 methylation also remained strongly associated with MSI in this model (OR = 6.3, 95% CI = 3.5–11.4, P < 10−8) but the inverse association between SFRP1 methylation and MSI was no longer significant (OR = 0.4, 95% CI = 0.1–1.2, P = 0.14).
Table IV.
Table IV.
Multivariate MSI analysis adjusted for age, sex, tumor location and DKK1 or SFRP1 methylation in Ontario and Newfoundland
Methylation and recurrence-free survival
To assess the relationship of DKK1 and SFRP1 methylation to patient outcome, recurrence-free survival analysis was performed. In Ontario, there were 120 recurrences and/or deaths due to CRC with a mean interval of 30 months from diagnosis. The mean follow-up time among remaining (censored) cases was 81 months. In univariate analysis, DKK1 methylation showed a trend toward an association with favorable outcome (HR= 0.6, 95% CI = 0.3–1.0, P = 0.06). Results were similar after adjustment for age, sex, stage and grade (HR = 0.5, 95% CI = 0.3–1.0, P = 0.06). SFRP1 methylation was not associated with outcome in Ontario.
In Newfoundland, there were 233 recurrences and/or deaths with a mean interval of 26 months from diagnosis. The mean follow-up time among remaining cases was 65 months. DKK1 methylation was not associated with outcome in univariate analysis (HR = 0.9, 95% CI = 0.6–1.3, P = 0.59) or in multivariate analysis adjusted for age, sex, stage and grade (HR = 0.9, 95% CI = 0.6–1.4, P = 0.62). As in Ontario, SFRP1 methylation was not associated with outcome in Newfoundland.
To assess the potential effects of MSI status on the association between methylation and survival, an MSI term was added to the multivariate survival models. In Ontario, the borderline association between DKK1 methylation and favorable outcome was lost (HR = 0.7, 95% CI = 0.3–1.3, P = 0.22), suggesting that the apparent benefit of DKK1 methylation may be due, at least in part, to its association with MSI. In Newfoundland, DKK1 and SFRP1 methylation remained unassociated with outcome.
We quantified promoter methylation of DKK1 and SFRP1 in a large cohort of MSI-stratified colorectal carcinomas from two distinct populations and examined associations between methylation status and patient clinicopathological characteristics. In both populations, DKK1 methylation was much more common in MSI tumors than MSS tumors and this association was independent of age, sex and tumor location. Conversely, in both populations, SFRP1 methylation was less common in MSI tumors and this inverse association with MSI was independent of age, sex and tumor location among Newfoundland probands. Collectively, these results suggest that DKK1 methylation is a robust marker of MSI CRCs, whereas SFRP1 methylation may be a marker of MSS tumors. More specifically, DKK1 methylation seems to be a marker of sporadic MSI but not Lynch syndrome-associated MSI as evidenced by its strong association to MLH1 methylation—the primary cause of sporadic MSI—but lack of association to germ line MMR mutation. This preference also indicates that DKK1 methylation is not a consequence of MSI per se.
We also examined associations to the BRAF V600E-activating mutation, a common oncogenic alteration in both MSI and CIMP tumors that has recently been proposed as a surrogate marker of CIMP (40)]. DKK1 methylation was strongly associated with BRAF V600E mutation in both populations. These associations remained significant after adjustment for MSI status suggesting that DKK1 methylation may also be a bona fide marker of CIMP. Importantly, the strong and independent associations of DKK1 methylation to both MSI and BRAF V600E indicate that DKK1 methylation may be a marker of MSI+CIMP+ tumors. Combined analysis of MSI and CIMP status has recently been proposed to help explain the substantial prognostic variation seen within the CIMP phenotype and MSI+CIMP+ tumors seem to be a favorable subset (41,42). This may highlight a role for DKK1 methylation as a clinically relevant prognostic marker. Concordantly, DKK1 methylation showed a borderline association with favorable recurrence-free survival in both Kaplan–Meier analysis and Cox regression analysis adjusted for age, sex, stage and grade but not after further adjustment for MSI status.
This is the first large study to show a difference in Wnt antagonist methylation between MSI subtypes in CRC. These findings add CRC to a growing list of cancers in which promoter methylation of Wnt antagonists has been associated to tumor subtype. Notably, SFRP1 methylation has previously been associated with MSI in a small series of endometrial tumors (43). Consequently, there seems to be some preference in epigenetic targeting of Wnt signaling in MSI-stratified CRC but these preferences may differ from those in other MSI-associated cancers.
The opposing associations of DKK1 and SFRP1 methylation to CRC MSI subtype may have important implications to subtype-specific tumor biology due to the differential contributions of these mediators to Wnt suppression. Although SFRPs are known to abrogate both the canonical and non-canonical Wnt pathways through interactions with Wnt ligands and/or Frizzled, DKKs seem to exclusively inhibit the canonical pathway by binding and internalizing the coreceptor LRP5/6 (reviewed in refs 44,45; Figure 1). Consequently, our results suggest that MSS tumors may experience preferential non-canonical Wnt activation through SFRP1 loss. Previous studies have identified MSI-dependent differences in canonical Wnt signaling by examining genetic, epigenetic and expression data (4648) but similar differences in non-canonical Wnt signaling have not been examined. Subtype-specific non-canonical Wnt activity may have important functional implications, as these pathways may promote invasion and metastasis (49,50) as well as potential clinical implications, as expression of non-canonical Wnt pathway components tends to associate with poor outcome in several cancers (4952). Interestingly, invasion and poor outcome are both hallmarks of MSS tumors, which may further implicate non-canonical Wnt activity as a possible contributor to subtype-specific biology and outcome in these CRCs.
Fig. 1.
Fig. 1.
Variation in the suppressive abilities of Wnt antagonists with respect to canonical and non-canonical Wnt signaling pathways. DKKs inhibit the canonical Wnt pathway by internalizing LRP5/6, whereas SFRPs inhibit both the canonical and non-canonical pathways (more ...)
The role of SFRP1 methylation as an indicator and contributor to this potential subtype bias in Wnt activity will require further scrutiny, including validation of the silencing role of methylation at the protein level in the primary tissue and how this affects downstream canonical and non-canonical Wnt activity. Furthermore, the association of DKK1 methylation with CIMP will require validation using a concerted CIMP panel. Nevertheless, the association of these events with opposing MSI subtypes paves the way for additional studies to examine subtype specificity among Wnt antagonists in hopes of revealing the prognostic and functional contributions of these events to cancer.
This work was supported by a Team Grant from the Canadian Institutes of Health Research (CTP-79845) awarded to J.R.M., B.B., J.A.K., S.S.G., R.C.G. and P.S.P.; by the National Cancer Institute (NCI) under Request For Applications (CA-95-011) and through cooperative agreements within the Colon Cancer Familial Registry (U01 CA074783) awarded to the Ontario Registry for Studies of Familial Colorectal Cancer (Principle Investigator: S.S.G.). The content of this article does not necessarily reflect the views or policies of the NCI or any of the collaborating centers in the Cancer Family Registry (CFR) nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. Government or CFR. J.B.R. was supported by graduate studentships from the Team in Interdisciplinary Research on Colorectal Cancer funded by the Canadian Institutes of Health Research and by graduate studentships from the Department of Laboratory Medicine and Pathobiology at the University of Toronto.
Supplementary material
Acknowledgments
The authors sincerely thank the investigators, staff and participants of the Colon Cancer Family Registry for their contributions to this project.
Glossary
Abbreviations
CIconfidence interval
CIMPCpG island methylator phenotype
CRCcolorectal cancer
DKK1-4Dickkopf
MMRmismatch repair
MSImicrosatellite instability
MSSmicrosatellite stable
NFCCRNewfoundland Familial Colorectal Cancer Registry
OFCCROntario Familial Colorectal Cancer Registry
ORodds ratio
PCRpolymerase chain reaction
SFRP1-5Secreted Frizzled Related Proteins
WIF-1Wnt Inhibitory Factor-1

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