1 Division of Human Nutrition and Epidemiology, Wageningen University, PO Box 8129, 6700 EV, Wageningen, The Netherlands and
2 Department of Pathology, UMC St Radboud, Nijmegen, The Netherlands
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Abstract |
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Abbreviations: APC, adenomatous polyposis coli; CI, confidence interval; MCR, mutation cluster region; OR, odds ratio; SSCP, single-strand conformation polymorphism
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Introduction |
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Although the etiology of colon cancer is complex and multifactorial, from the perspective of understanding the genetic events involved in carcinogenesis, it is one of the best-characterized epithelial tumors. Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, i.e. those resulting in loss of APC function, are thought to be a key initiating event in familial as well as in sporadic colorectal cancer. They can be detected in many sporadic adenomas and carcinomas, including adenomas as small as 5 mm in diameter (2). Recently, both Fodde et al. (3) and Kaplan et al. (4) showed that APC is also involved in chromosomal segregation and that truncation of APC causes chromosomal instability in embryonic stem cells. This suggests that loss of APC function is not only important for tumor initiation but may play a role in later stages of malignant progression as well.
In contrast to the germline mutations, which are scattered over a large part of the gene, the majority of the somatic APC mutations seem to cluster within a small region in exon 15 (codon 12861513), the so-called mutation cluster region (MCR) (57). Truncating mutations (that is, nonsense and frameshift mutations) in the MCR have been reported to occur in 3045% of the sporadic colon tumors (2,5,6,8). The observed clustering of somatic mutations in APC could be caused by hypermutability of this specific region, a selective advantage for tumor formation exerted by mutations in this region, or a combination of these two. Results from several studies indicate that truncating mutations in the MCR indeed provide cells with a selective growth advantage, probably due to inactivation of the ß-catenin down-regulating function of APC (9,10).
The relationships between environmental factors and the genetic and epigenetic [e.g. DNA methylation, see refs (11,12)] alterations that drive colon carcinogenesis are not (yet) clear. However, dietary factors reported previously to be associated with colon cancer risk may well, directly and/or indirectly, influence the occurrence of somatic truncating APC mutations in colon tumors. Bardelli et al. (13) demonstrated recently that exposure to specific carcinogens can indeed select for tumor cells with distinct forms of genetic instability. Dietary factors have been found associated with the occurrence of specific K-ras mutations in human colon carcinomas (14,15). Moreover, the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), an in cooked meat abundantly present heterocyclic amine, has been linked to specific Apc mutations in rat colon tumors (16,17).
Therefore, in this study, we assess the associations between specific dietary factors (all reported previously to be associated with colon cancer risk) and the occurrence of truncating mutations in the MCR region of APC in a population-based case-control study of incident cases of sporadic colon carcinomas. This is, to our knowledge, the first study that examines these associations in a human population.
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Materials and methods |
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Data collection
Usual dietary habits were assessed by an interview-based questionnaire. The questionnaire covered the complete dietary pattern. The interval between diagnosis and interview was, for cases, 36 months. The consumption frequency per month in the preceding year (for cases, the year preceding diagnosis or symptoms), number of months during which the item was used, number of portions per consumption and portion sizes of 289 food items were collected. To check for internal consistency, the consumption frequencies of various meal components were compared with the total meal pattern. Frequently used household utensils and cups were weighed to be able to estimate portion sizes. Average daily intake of nutrients was calculated using the Dutch National Food Table (19). The interviewing of cases and controls was balanced over seasons to account for seasonal fluctuations in food patterns. During the interview, information was also obtained on current and previous smoking habits, aspirin and non-steroidal anti-inflammatory drug use, family history of colorectal cancer and medical history, see also Kampman et al. (20).
APC mutation detection
DNA was isolated from tumor-rich areas (>60% tumor cells) as described elsewhere (21). Single-strand conformation polymorphism (SSCP) analysis was used to screen the APC gene for nonsense and frameshift mutations. Most studies on somatic mutations in APC focus on the MCR of the gene (codons 12861513). Our analysis covers codons 12861585 (extended-MCR) of APC and includes codon 1554 in which somatic mutations are also often observed (7) (APC database: http://perso.curie.fr/Thierry.Soussi/APC/html). The region was divided into five, 220 bp long, overlapping fragments (codons 12861358, 13371404, 13871455, 14371526 and 15091585, respectively) which were amplified separately in two consecutive PCRs using the following primer sets (primer sequence: 5'
3'):
Fragment 1:codon
1.1 F-CAGACTTATTGTGTAGAAGR-CGCTCCTGAAGAAAATTCAAG12601358
1.2 F-GAAATAGGATGTAATCAGACGR-CGCTCCTGAAGAAAATTCAAC12861358
Fragment 2:
2.1 F-ACTGCAGGGTTCTAGTTTATCR-TCTGCTTGGTGGCATGGTTT13371436
2.2 F-ACTGCAGGGTTCTAGTTTATCR-GAGCTGGCAATCGAACGACT13371404
Fragment 3:
3.1 F-CTCAGACACCCAAAAGTCCR-ATTTTTAGGTACTTCTCGCTTG13661455
3.2 F-TACTTCTGTCAGTTCACTTGATAR-ATTTTTAGGTACTTCTCGCTTG13871455
Fragment 4:
4.1 F-AAACACCTCCACCACCTCCR-TCATTCCCATTGTCATTTTCC14371536
4.2 F-AAACACCTCCACCACCTCCR-GCATTATTCTTAATTCCACATC14371526
Fragment 5:
5.1 F-ACTCCAGATGGATTTTTCTTGR-GGCTGGCTTTTTGCTTTAC14971596
5.2 F-GAGCCTCGATGAGCCATTTAR-TGTTGGCATGGCAGAAATAA15091585
PCR reaction mixtures (total volume 50 µl) contained 50 ng DNA or 2 µl of the 1:100 diluted product of the first PCR, 0.2 µM of both primers, 0.2 mM dNTPs, 10 mM TrisHCl pH 9.0, 1.52.5 mM MgCl2, 50 mM KCl, 0.01% Tween, 10% glycerol and 0.3 U Taq DNA polymerase. Reaction conditions first PCR: 25 cycles of 30 s at 94°C, 45 s at 55°C (53°C for primer set 1.1; 57°C for primer set 4.1), 1 min at 72°C, followed by 5 min at 72°C. Reaction conditions second PCR: 30 cycles of 30 s at 94°C, 45 s at 52°C (56°C for primer sets 3.2 and 4.2; 57°C for primer set 5.2), 1 min at 72°C, followed by 5 min at 72°C. Products were checked using an ethidium bromide stained 2% agarose gel. SSCP was performed as described earlier (21) with electrophoresis at 10 and 18°C. The original PCR products from the samples that displayed an abnormal pattern in the SSCP were subjected to sequencing in both directions using the same primers as in the second PCR. Sequencing was performed as described previously (21). Mutation analysis started in all samples with fragment 1 and only if no nonsense or frameshift mutations (resulting in truncated, non-functional APC protein) were detected fragment 2 was screened for mutations, and so on.
Statistical analyses
Cases were classified as APC+ (carcinomas containing a nonsense or frameshift mutation in the extended-MCR of APC), or APC- (carcinomas without a nonsense or frameshift mutation in the extended-MCR of APC). Energy-adjusted nutrient intakes were computed, for women and men separately, as the residuals from the regression model with total energy as the independent variable and absolute nutrient intake as the dependent variable. As a constant, the mean of the nutrient intake was added to each residual (22,23). Differences in characteristics between the groups were assessed using t-tests for continuous and 2 tests for categorical variables. The categorization of food groups and nutrients, in tertiles, was based on the distribution of intake in the control population. To look at the combined effect of vegetable consumption and red meat intake, mutually exclusive categories of combinations of the two exposures were defined based on median split. Case-control comparisons, separately comparing APC+ cases and APC- cases with the population-based controls, were conducted to estimate the relative risk of developing carcinomas, respectively, with and without a truncating APC mutation. Case-case comparisons were conducted to evaluate heterogeneity in dietary risk factors for the two subsets of carcinomas. Odds ratios (ORs) and the corresponding 95% confidence intervals (95% CI) were calculated using multiple logistic regression models. Linear trend was assessed using the tertile medians as continuous variables in multiple logistic regression models. To quantify the associations on a continuous scale and allow direct comparisons between the different vegetable subgroups, ORs and 95% CIs were also calculated for a fixed amount (10 g/day) of intake (24). All analyses were adjusted for age, sex and total energy intake. Alcohol intake was additionally adjusted for smoking. Additional adjustment for Dukes stage, tumor location, smoking, body mass index and other dietary factors, did not change the estimates significantly (that is, not more than 10%). Analyses were performed with the use of the SAS® Statistical Software Package (SAS version 6.12; SAS Institute, Cary, NC).
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Results |
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Characteristics of the study population are given in Table I. Cases were divided into two groups: APC+, individuals with a tumor exhibiting a truncating APC mutation; and APC-, individuals with a tumor without a truncating APC mutation. Age, sex and body mass index did not differ significantly between cases and controls, and neither between APC+ and APC- cases. Total energy intake was higher among the cases, and highest among the APC- cases. The frequency of Dukes stage C and D tumors did not differ significantly between the two case groups, proximal tumors (caecum, ascending colon, hepatic flexure and transverse colon) were slightly more common among the APC- cases. There were more ever smokers in the APC- group than in the APC+ group.
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Table II presents results of case-control and case-case comparisons conducted to assess associations between the various food groups and APC+ and APC- tumors. Total vegetable intake was inversely associated with APC+ tumors (although here the association was not statistically significant) as well as APC- tumors when the two tumor subsets were separately compared with the population-based controls. Interestingly, case-case comparison showed that total vegetable intake was significantly different related to APC+ tumors than to APC- tumors. Similar patterns of association were observed for the evaluated vegetable subgroups: leafy greens, cruciferous, allium and root vegetables. To allow direct comparisons between the different vegetable subgroups and to quantify associations on a continuous scale, ORs and 95% CIs were also calculated for a fixed amount (10 g/day) of intake. The strongest inverse associations were observed for allium vegetables and leafy greens with APC- tumors (APC- versus controls, OR: 0.81, 95% CI: 0.700.94; OR: 0.86, 95% CI: 0.760.97, respectively; not in table). Additional adjustment for meat consumption did not alter the observed associations significantly.
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To look at the combined effect of vegetable and red meat consumption, mutually exclusive categories of combinations of the two exposures were defined based on median split. Individuals with high meat (72 g/day)/low vegetables (<191 g/day) intake showed a substantially increased risk for APC+ tumors (APC+ versus controls, OR: 2.9, 95% CI: 1.27.2) as well as APC- tumors (APC- versus controls, OR: 2.1, 95% CI: 1.14.2) compared with individuals with low meat (<72 g/day)/high vegetables (
191 g/day) intake (not in table).
Assessment of nutrients (presented in Table III) showed that alcohol intake was positively associated with APC- tumors whereas an inverse, statistically non-significant, association was observed with APC+ tumors. Moreover, case-case comparison demonstrated that alcohol was significantly different related to APC+ tumors than to APC- tumors. Total fat was positively associated with APC+ tumors, and cases with high total fat intake do appear to more likely develop an APC+ tumor than an APC- tumor (Table III
). Interestingly, no clear associations were observed for saturated fat but unsaturated fat was strongly, positively, associated with APC+ tumors. Cholesterol was positively associated with both tumor groups, most pronounced and significantly with APC- tumors. Carbohydrate was inversely associated with both tumor groups, most pronounced and significantly with APC- tumors. Dietary fiber, vitamin C and ß-carotene showed association patterns similar to those observed for vegetable consumption. However, all three are related to vegetable intake and after inclusion of total vegetables in the multivariate model, the observed ORs were attenuated and dietary fiber was no longer significantly different related to APC+ tumors than to APC- tumors (data not shown).
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Discussion |
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We focused on nonsense and frameshift mutations in the MCR because these mutations indisputably result in truncated and non-functional APC protein whereas the biological significance of missense mutations in APC is uncertain. Allelic loss at APC also results in loss of APC function. At the moment, however, allelic loss is thought to be a primarily spontaneous event (25). Besides that, colon tumors in which APC is mutated usually show allelic loss plus a truncating mutation in the MCR or two truncating mutations (at least one in the MCR) but rarely only allelic loss or only mutations outside the MCR (7). We therefore do not expect that our decision to not consider allelic loss and to concentrate on truncating mutations in the MCR has resulted in extensive misclassification.
Using SSCP analysis and sequencing, we identified truncating mutations in the extended-MCR (codons 12861585) of APC in 34.2% of the carcinomas. Despite the fact that our frequency seems low when compared with the conventional wisdom that most colon tumors follow a genetic pathway involving APC, it is consistent with the mutation frequencies for this region, 3045%, reported by most others (2,5,6,8). Only Rowan et al. observed, in tumor cell lines, a higher frequency, 60% (7). The characteristics of the mutations and the hotspots we identified were also similar to those observed by others (6,7) (APC database: http://perso.curie.fr/Thierry.Soussi/APC/html). Although microdissection was performed, it remains possible that, due to contaminating normal tissue, mutations were missed eventually resulting in misclassification, i.e. tumors with a truncating mutation in the APC- group. This may have attenuated some of our results.
The case-control study was initially designed to examine the role of dietary factors in the etiology of sporadic colon cancer in general (that is, colon cancer not categorized according to mutational status of the tumors). The results of the cases versus controls comparisons, reported previously by Kampman et al. (18,20), were in line with those reported by others (1). As in any retrospective study, information bias and selection bias may have affected our results. Cases and controls were asked to recall their diets from the past and differential recall is possible. One of the advantages of the conducted case-case comparisons is, however, that the cases are unaware of the mutational status of their tumors. Consequently, systematic errors in dietary recall are less likely to bias results from case-case comparisons. Recall of dietary habits can also be influenced by tumor stage or treatments that affect appetite. Our cases were relatively healthy. That is, the frequency of Dukes A and B tumors among the cases was relatively high, 63%, compared with the frequency reported by the Dutch Cancer Registry, 51% (26). Adjusting the case-case comparisons for Dukes stage did not change the estimates significantly.
We calculated the largest and smallest OR detectable with a power of 0.90 for the four different study populations used here in order to determine what effects we were able to exclude (27). For all cases versus controls, the present study is able to detect ORs 0.5 and
1.9; for APC+ versus controls,
0.3 and
2.5; for APC- versus controls,
0.4 and
2.0; and for APC+ versus APC-,
0.3 and
2.7. Although the dietary factors evaluated in this study were all previously identified risk factors for colon cancer, it should be noted that multiple comparisons might lead to chance findings.
Dietary factors may influence the occurrence of truncating APC mutations in colon carcinomas directly, i.e. by being involved in the actual production or prevention of these mutations, and/or indirectly, i.e. by being involved in the promotion or evasion of progression into later stages and eventually into carcinomas. In this study, we observed a protective effect of vegetables for APC+ tumors as well as APC- tumors. Interestingly, the protective effect was markedly higher for APC- tumors. So far, to our knowledge, no other studies have evaluated dietary factors and APC mutations in human colon carcinomas. However, in line with our results, low vegetable intake was reported to also increase the risk of colon carcinomas with and without a K-ras mutation in a large study on diet and K-ras mutations in sporadic colon carcinomas. And, there too, the increase was most pronounced for tumors without a mutation (15).
A possible explanation for the observed difference in protective effect of vegetables between the two tumor subsets is that, as also suggested by other observations reported previously (3,4,28), loss of APC function is not only important for tumor initiation but plays a role in later stages of malignant progression as well. Hence, the protective influence of vegetables on progression might be more limited after an APC mutation has occurred than in otherwise initiated tumors. The latter appear to follow a different pathway (29) and possibly need to undergo more and/or different genetic and epigenetic changeswhich may be prevented by vegetablesbefore they eventually are able to develop into carcinomas.
Also, alcohol was significantly different related to APC+ tumors than to APC- tumors in our population. It increased the risk of APC- tumors but not of APC+ tumors, which points to a (negative) role in the pathway of tumors without a truncating APC mutation in particular. Alcohol possibly exerts its effect on colon cancer through interference with folic acid availability (3032). Folate is involved in DNA methylation and appears to be essential for normal DNA synthesis and repair (33). Vegetables, especially green leafy and cruciferous vegetables, contain large amounts of folate and are the main dietary sources. Disturbances in DNA methylation pathways can result in chromosome breaks due to deficient methylation of uracil to thymine (34), but also in epigenetic silencing of genes (11,12). Regarding the latter, hypermethylation of the promotor region of the hMLH1 geneobserved in the majority of sporadic colon carcinomas with microsatellite instabilityseems to be particularly important for tumor development as it results in the inactivation of the DNA mismatch repair system (3537) which, in turn, can lead to the additional loss of other genes involved in carcinogenesis due to repairs not executed. Inverse relationships have been reported between microsatellite instability and mutations in APC and p53 which suggests that APC+ tumors and tumors with microsatellite instability develop through different pathways (38). Thus, microsatellite instability positive tumors are probably most common in our APC- group. Interestingly, and consistent with our results, Slattery et al. (39) recently reported that long-term alcohol consumption increased the probability of developing sporadic colon carcinomas with microsatellite instability.
For animal products, no statistically significant associations were observed. However, red meat and fish did seem more notably associated with APC+ tumors than with APC- tumors, which suggests that products of animal origin may influence the occurrence of truncating APC mutations in colon carcinomas positively. Red meat and fish, prepared at high temperatures, are major sources of heterocyclic amines (40). Heterocyclic amines are bulky-adduct-forming agents that can produce DNA strand breaks (41) and probably so contribute to carcinogenesis. They have been shown to be carcinogenic in rodents (42,43) and to induce specific deletion mutations in the Apc gene in rat colons (16,17). High intake of certain heterocyclic amines was also found associated with increased risk of colorectal adenomas in humans (44).
Interestingly, we observed that fat, especially unsaturated fat, increased the risk of APC+ tumors in our population. Recent studies on specific fatty acids and colon cancer (in general) have reported no clear association with colon cancer risk (45,46). However, intake of unsaturated fat (especially linoleic and 20-carbon poly-unsaturated fatty acids) does appear to be more strongly associated with colon cancer risk among those with a family history of colorectal cancer than among those without (45). Slattery et al. (15) also observed that fat (mono-unsaturated fat most pronounced) was related differently to carcinomas with a GT transversion at the second base of K-ras codon 12 than to tumors without a K-ras mutation. In our own population, as reported earlier (14), we did not observe an association between dietary fat and K-ras mutations.
In summary, our data suggest that vegetables play a protective role in the etiology of APC+ tumors as well as APC- tumors, although the protective effect of vegetables appears to be less influential in the APC+ subset. Alcohol seems to promote the development of APC- tumors in particular whereas meat, fish and fat appear to enhance the development of APC+ tumors. This supports the idea that APC+ tumors and APC- tumors develop through different pathways affected by specific dietary factors. Our results, if confirmed in other studies, provide further clues to the relationships between dietary factors and the molecular alterations that drive colon carcinogenesis.
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Notes |
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Acknowledgments |
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References |
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