1 Department of Toxicology Pathology and Genetics, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands, 2 Department of Pathology, Josephine Nefkens Institute, Erasmus University Rotterdam, Rotterdam, The Netherlands and 3 Division of Human Nutrition, Wageningen University and Research Centre, Agrotechnion, Bomenweg 2 (bode 62), 6703 HD Wageningen, The Netherlands
4 To whom correspondence should be addressed Email: ellen.kampman{at}wur.nl
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: AA, arachidonic acid; 95% CI, 95% confidence intervals; COX, cyclooxygenase; cPLA2, cytosolic PLA2; EPA, eicosapentaenoic acid; FAP, familial adenomatous polyposis; LOX, lipoxygenase; ORs, odds ratios; PPAR, peroxisome proliferator-activated receptor; PUFAs, polyunsaturated fatty acids; sPLA2, secretory PLA2; SNPs, single nucleotide polymorphisms; 3'-UTR, 3'-untranslated region
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclooxygenases (COX) 1 and 2, also known as prostaglandin endoperoxide synthases, are two key genes in the AA pathway, encoding enzymes that initiate the synthesis of biologically important prostanoids (PGs) and eicosanoids (2). Both genes have been demonstrated to be involved in intestinal tumorigenesis, by promoting tumor growth, angiogenesis and metastasis. Major evidence comes from the study of non-steroidal anti-inflammatory drugs, acting amongst others as COX inhibitors, which play an important role in cancer prevention (3). Inactivation of these genes in mouse models of intestinal cancer decreases the number of polyps in these mice (4,5). Phospholipases A2 are an enzyme family that, besides other conversions, catalyze the generation of free fatty acids, such as AA, from membrane bound phospholipids. Most important in the AA pathway are cytosolic phospholipase A2 (cPLA2) and secretory phospholipase A2 (sPLA2), which have both been demonstrated to be involved in tumorigenesis in mouse models (6,7). Levels of cPLA2 mRNA are reduced in mouse and human tumors, possibly indicating a tumor protective effect of this enzyme, which is further substantiated by the reduction in apoptosis in vitro after inhibiting cPLA2 (6). A similar protective effect is found for the secretory form of the enzyme, sPLA2. This gene, present at the Mom1 locus, causes an increase in the number of polyps when inactivated in the ApcMin mouse model of intestinal cancer (8). Lipoxygenase (LOX) is an enzyme for which AA is one of the substrates. Activation of the enzyme 15LOX might inhibit carcinogenesis via the conversion of linoleic acid into 13-S-hydroxyoctadecadienoic acid, which in turn down-regulates peroxisome proliferator-activated receptor (PPAR) , thereby restoring apoptosis (9). PPARs play an important regulatory role in lipid metabolism and cancer and PPARs can be activated by a variety of eicosanoids (10,11). There are three distinct types of PPARs,
,
and
. Both PPAR
and PPAR
have been implicated in colorectal tumorigenesis, by transcriptionally controlling pathways involved in cell proliferation, differentiation and survival. Activation of PPAR
was shown to increase the number and size of intestinal polyps in ApcMin mice (12), whereas inactivation causes inhibition of tumor growth in a colorectal cancer xenograft model (13,14). Moreover, a role as a focal point of cross-talk between prostaglandin and Wnt signalling pathways has recently been suggested (15). Most data on PPAR
suggest a tumor suppressive role, however, there is still some controversy about the increase in intestinal polyps in ApcMin mice by ligands of PPAR
(16,17).
Genetic variants represented by single nucleotide polymorphisms (SNPs) in genes encoding these key players of the AA pathway may contribute to variation in susceptibility to colorectal cancer. Recently the focus of attention has shifted from the use of single (putative causal) genetic variants in association studies (the direct approach) to using sets of genetic markers without a priori functional effects, the so-called indirect approach (18). Using information about the common SNPs in a particular population, combinations of SNP alleles called haplotypes can be estimated, after which differences in frequencies between cases and controls can be evaluated (19,20).
Colorectal adenomatous polyps, later referred to as colorectal adenomas, are presumed to be the precancerous state of colon cancer (2). Studying colorectal adenomas instead of colon cancer may give information about the risk factors in the earlier stages of carcinogenesis.
The fatty acids utilized by the AA pathway include n-3 polyunsaturated fatty acids (PUFAs) from fish. Some, but not all, animal experimental studies and epidemiological studies have shown that fish consumption may decrease the risk of colorectal tumors (21,22). A high ratio of fish fatty acids to AA in adipose tissue, as a marker for fatty acid intake, has been associated with a lower risk of colorectal adenomas (23). High fish consumption can affect the AA pathway by causing a shift in substrates from n-6 to n-3 PUFAs. SNPs in AA pathway genes may interact with fish consumption by influencing the conversion of these PUFAs into eicosanoids.
In this association study 21 SNPs in genes encoding five enzymes and two nuclear receptors have been used. The objective of this study was to assess the association between inherited SNPs and haplotypes involved in the AA pathway and the occurrence of colorectal adenomas. Moreover, the potential modifying effect of fish consumption was considered.
![]() |
Methods and materials |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Major indications for endoscopy among cases were complaints (47.7%), including bowel complaints, rectal bleeding and defecation problems, and screening (39.5%). For controls these numbers were 76.7% for complaints and 1.7% for screening.
Pathological anatomy reports provided information about polyp characteristics. Medical records were used for additional information on polyp recurrence and general health status of the participants.
The total study population included 925 subjects. The Medical Ethical Committees of all participating hospitals and of Wageningen University approved the study and all participants have provided written informed consent.
Genetic analysis
Twenty of the 21 SNPs were selected on the basis of an inventory of genetic variation in the Dutch population of the selected genes as described elsewhere, in which 58 polymorphisms were identified (C.Siezen, in preparation). The SNP selection was based on allele frequency (with some exceptions, only those SNPs with a minor allele frequency of 5% or higher were considered), position in the gene (when possible divided over the length of the gene), possible impact on protein function (amino acid changes) and linkage between the SNPs in one gene (of two or more tightly linked SNPs only one was selected). One SNP was selected on the basis of another population study on COX-1 variants (26).
Genotypes of the 21 SNPs were determined using a technique known as PyrosequencingTM. Each PCR contained 5 µl of 2x Hotstar master mix (Qiagen), 1 µM first primer, 0.1 µM second primer containing a so-called universal tail of 23 nt, 0.9 µM third primer with the same sequence as the tail and labeled with biotin and 10 ng genomic DNA, in a total volume of 10 µl. PCR reactions were carried out in a Perkin-Elmer 9600 thermal cycler under the following conditions: 95°C for 15 min; 40 cycles of 94°C for 45 s, 57°C for 45 s and 72°C for 1 min; 72°C for 10 min. From the biotinylated PCR products single-stranded DNAs were prepared and subsequently genotyped using a PSQ 96MA system (Pyrosequencing AB) (http://www.pyrosequencing.com) and SNP reagent kit (Pyrosequencing AB), as previously described (27). Primers for each SNP are described in the Appendix, Table AI. DNA was available from 808 participants. SNPs could not be assessed in one of these samples.
|
Information on demographic and lifestyle factors, like smoking habits, physical activity level (30) and family history, was obtained from a self-administered questionnaire.
The intake of total energy and of various nutrients was calculated from this questionnaire using the computerized Dutch food composition table.
Statistical analysis
Subjects with incomplete dietary data (n = 20) were excluded. The data analysis thus included data for 787 participants: 384 cases and 403 controls.
Logistic regression analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (95% CI) of separate genotypes when possible. If the numbers were too small, analyses were performed using pooled heterozygous and homozygous minor genotypes. The only potential confounding variables included in the model were age and gender.
Haplotypes were estimated and ORs calculated using the Hplus program, available online at http://qge.fhcrc.org/hplus. Hplus is a SNP analysis tool for performing haplotype estimations according to the distribution of genotypes in a population. It is able to handle datasets that include casecontrol status as well as covariates and SNP location variables (31).
Multiple logistic regression analysis was performed to evaluate the modifying effect of fish consumption. Fish consumption, in grams per day, was divided into tertiles according to the distribution of intake among controls. The lowest fish consumption tertile in combination with a homozygous major allele for the SNP of interest was considered as the reference group.
The variables age, body mass index, family history of colorectal cancer, gender, indication of endoscopy, insulin use, physical activity, education level, smoking, aspirin use, daily energy intake and intake of alcohol, calcium, fiber, fruit, red meat, poultry, processed meat, vegetables, ß-carotene, vitamin C and vitamin E were considered as potential confounding factors. Besides the variables age and gender, indication for endoscopy and alcohol consumption were also included in the model, since these factors changed the ß estimates by >10%.
To test for linear trend, we modeled the tertile of fish consumption as a continuous variable in the logistic regression model, in which each tertile was assigned its median value. To test whether the combination of genotypes and fish consumption deviated from multiplicativity, we calculated P values for interaction by inclusion of a numerical term for genotype, multiplied by fish consumption as a continuous variable, into our multivariate models. To exclude the influence of previous adenomas among cases and of undetected proximal polyps among controls, we repeated our analysis excluding these cases and controls.
The analyses were conducted using Statistical Analysis Software (SAS) for Windows, version 8.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genotypes
Table I shows the associations between genotypes and colorectal adenomas. A statistically significant inverse association was observed for the CT genotype of SNP H477H (OR 0.63, 95% CI 0.450.89) in the PPAR gene as compared with the CC genotype. The OR of the TT genotype did not reach significance, but the TC and TT genotypes together also showed a statistically significant inverse association (OR 0.64, 95% CI 0.460.90) (data not shown) as compared with the CC genotype. For the COX-2 gene two opposite associations were observed; an inverse association for the GC genotype of SNP V102V (OR 0.65, 95% CI 0.460.92) and a positive association for the TC genotype of SNP c.2242T
C (OR 1.47, 95% CI 1.072.00) as compared with the homozygous major genotypes. In the case of both SNPs the homozygous minor genotypes did not show a statistically significant OR, but combining the heterozygous and homozygous minor genotypes resulted in a statistically significant association (OR 0.68, 95% CI 0.490.95 and OR 1.40, 95% CI 1.041.89, respectively) (data not shown), as compared with the homozygous major genotypes.
|
Haplotypes
Table II shows the associations between the most common haplotypes estimated and colorectal adenoma risk. The haplotypes are represented as a series of 0s and 1s, indicating for each SNP in that gene whether the major (0) or minor (1) allele is present in that haplotype. Five statistically significant associations between a specific haplotype and adenoma risk have been found. A reduction in the risk for colorectal adenomas was observed for haplotype 01 in PPAR (C allele for P12A and T allele for H477H) and 11 in 15LOX (C allele for c.-217G
C and G allele for T485T). The ORs showed an
50% reduction in colorectal adenoma risk (OR = 0.47 and 0.51, respectively), as compared with haplotype 00 (major alleles for both SNPs). The remaining three haplotypes showed a positive association. Haplotypes 100 and 001 in COX-2 (G alleles at position c.-1329 and amino acid 102 and the T allele for c.2242T
C and A allele at position c.-1329, G allele at amino acid 102 and C allele for c.2242T
C) showed similar ORs of 1.37 (95% CI 1.011.86) and 1.34 (95% CI 1.021.76), as compared with haplotype 000. An even greater effect was observed for haplotype 001 in sPLA2 (C allele for c.-180C
G, G allele for T32T and T allele for c.665C
T), with an OR of 2.10 (95% CI 1.113.95), as compared with haplotype 000.
|
|
The AA genotype of SNP R651K, an AG substitution in cPLA2, was inversely associated with colorectal adenomas for individuals in the highest fish consumption tertile (OR 0.64, 95% CI 0.410.96), as compared with low fish consumption. The risk of colorectal adenomas also decreased for the GG genotype of SNP c.2605G
A in cPLA2 with high consumption of fish (OR 0.59, 95% CI 0.370.92), as compared with low consumption of fish.
After stratification for fish consumption, no statistically significant associations were observed between genotype and adenomas for either SNP in PPAR or for the SNPs in COX-1. There was also no effect on the associations between the genotypes of SNPs in sPLA2 and 15LOX and colorectal adenomas.
A summary of the major findings of this study is provided in Table IV.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The associations found between SNPs in COX-2 and colorectal polyps are supported by similar findings by Campa et al. (32), who found a positive association between the minor allele of SNP c.2242TC in COX-2 and non-small cell lung cancer. This is not, however, in line with a previously published study in which affected siblings were tested for linkage of COX-2 variants with colon neoplasia (33). The authors suggested that genetic variation in COX-2 is unlikely to contribute to colorectal cancer risk, but, as pointed out by Ulrich and Potter (34), the population used in this study might not be representative of the general population and markers some distance away from the COX-2 locus were used. Moreover, the importance of the 3'-UTR of COX-2 is illustrated by a strong positive association between another SNP in this region and colorectal cancer observed in a study investigating a number of polymorphisms in COX-2 (35). This part of the 3'-UTR was previously thought to lie outside the gene, hence this SNP was not included in our study. In line with published data on the importance of the 3'-UTR in post-transcriptional regulation of COX-2 expression (36) it is hypothesized that SNPs in this region interfere with this process.
We found no associations between the two SNPs in COX-1 and colorectal adenomas. This is in line with two previous studies investigating one of the SNPs in COX-1 (L237M), neither of which showed a significant association (26,37). However, due to the low minor allele frequency of this SNP, false negative results cannot be ruled out.
As far as we know, H477H in PPAR has not been tested before for colorectal tumor risk, whereas for the other variant in PPAR
, P12A, a modest protective effect of the minor allele has been found (38). This finding could not be replicated by our study, which may be explained in part by the different study populations, colorectal cancer versus adenoma patients. The involvement of this SNP in colorectal cancer development was also implicated by a study on colorectal cancer patients where the minor allele was frequently found in tumors without a K-ras mutation (39), an observation that could not be reproduced by us (data not shown). Analyzing the haplotype containing the major allele of P12A and the minor allele of H477H strengthened the association we found between H477H in PPAR
and adenomas. Moreover, the importance of specific haplotypes containing these SNPs was also illustrated by two other studies, on body weight and type 2 diabetes. However, the statistically significant association in the second study concerned the haplotype containing the minor allele of P12A and the major allele of H477H (also known as C1431T) (40,41). This does indicate, however, that there is likely a functional effect resulting from the combination of these two SNPs, either on protein function or on the amount of protein present.
We found a positive association between haplotype 001 (C allele for c.-180CG, G allele for T32T and T allele for c.665C
T) of sPLA2 and adenomas. When the SNPs were analyzed separately no associations were apparent, illustrating again the importance of specific haplotypes. This positive association is particularly striking since the role of sPLA2 in human colorectal carcinogenesis following the discovery of the gene at the Mom1 locus (42) has been the subject of much debate. Although one study found a germline sPLA2 mutation in a sporadic colorectal cancer patient (43), there have been several other results questioning the tumor-suppressing role of human sPLA2. No somatic mutations in tumors have been found (44), neither do the differing disease phenotypes in familial adenomatous polyposis (FAP) patients correlate with sPLA2 mutations (45). Gene expression analysis revealed no change in expression levels of sPLA2 between normal mucosa and tumors (46) and an apparent lack of expression of sPLA2 was observed in colorectal cancer cell lines (47). In line with this, a study investigating the effect of single SNPs, corresponding to two of our selected SNPs (c.-180C
G and T32T), on the phenotype of FAP patients and on sporadic colorectal cancer found no associations (48). However, the effect of the third SNP (c.665C
T) and the corresponding haplotypes was not considered in that study. Our results reinforce the notion that sPLA2 is not only an important tumor suppressor in mice, but may also play a role in human tumors.
No previous studies have been reported evaluating associations between SNPs or haplotypes in the anticarcinogenic 15LOX gene and colorectal tumors. However, associations with a specific haplotype in another subtype of the LOX enzyme, the procarcinogenic 5LOX gene, have been reported recently (37), again indicating the importance of this family of enzymes in colorectal tumorigenesis.
Fish consumption can affect lipid metabolism through different mechanisms. An increase in n-3 PUFAs can lead to an increase in eicosapentaenoic acid (EPA) tissue content, and a decrease in n-6 PUFA-derived AA. This will lead to an increase in eicosanoid synthesis from EPA, resulting in a shift in production of 2-series to 3-series prostaglandins (49,50). Since prostaglandins act as ligands for PPAR (51), fish consumption may interact with PPAR
by modifying the spectrum of PPAR ligands. This can be influenced by SNPs, explaining the interactions found between SNPs in PPAR
and fish consumption. Besides changing the substrates and therefore the products of the AA pathway, n-3 PUFAs can also have a direct effect on the genes in the pathway. A high concentration of n-3 PUFAs has been shown to inhibit COX-2 directly, causing a decrease in the overall production of prostaglandins (52). SNPs in COX-2 might interfere with this process. Inhibition of
6-desaturase, the rate-limiting enzyme in the conversion of linoleic acid to AA, by n-3 PUFAs has also been demonstrated (53). These interactions between fish consumption, as a proxy for n-3 PUFAs, and genes in the AA pathway make it plausible that the association between SNPs in these genes and colorectal adenomas is modulated by fish consumption and, therefore, that some associations became apparent and others were strengthened, although the exact mechanisms remain far from clear.
Consideration must be given to the potential limitations of the present study, in particular the possibility of chance findings due to evaluating a large number of genes and genediet interactions simultaneously. Although the genes examined have been previously shown to be involved in colorectal carcinogenesis, the results of this study need to be confirmed by others.
In conclusion, this study has shown that polymorphisms in genes involved in the AA pathway may be associated with colorectal adenomas. We have shown for the first time that these associations could be modified by fish consumption, but further research to understand the mechanisms involved is needed. For example, functional studies of the SNPs implicated in this study might provide a plausible basis for the associations observed and, to minimize information bias and to assess whether n-3 fatty acids are the active agents associated with colorectal tumor risk, measurements of n-3 fatty acids in plasma samples are recommended, and are presently ongoing.
![]() |
Appendix |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|