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Nutrition and Disease

Dietary Folate and APC Mutations in Sporadic Colorectal Cancer¹

² Research Institute Growth and Development (GROW), Department of Epidemiology; ³ Research Institute Growth and Development (GROW), Department of Pathology; and ⁴ Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Epidemiology, Maastricht University, Maastricht, The Netherlands and ⁵ TNO Quality of Life, Business Unit Food and Chemical Risk Analysis, Zeist, The Netherlands

^* To whom correspondence should be addressed. E-mail: stefan.devogel{at}epid.unimaas.nl.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Folate deficiency has been associated with colorectal cancer risk and may be involved in colorectal carcinogenesis through increased chromosome instability, gene mutations, and aberrant DNA methylation. Within the Netherlands Cohort Study on diet and cancer, we investigated the associations between dietary folate intake and colorectal cancer risk with (APC⁺) and without (APC^–) truncating APC mutations, accounting for hMLH1 expression and K-ras mutations. In total, 528 cases and 4200 subcohort members were available for data analyses of the study cohort (n = 120,852) from a follow-up period between 2.3 and 7.3 y after baseline. Adjusted gender-specific incidence rate ratios (RR) over tertiles of folate intake were calculated in case-cohort analyses for colon and rectal cancer. Although relatively high folate intake was not associated with overall colorectal cancer risk, it reduced the risk of APC^–colon tumors in men (RR 0.58, 95% CI 0.32–1.05, P_trend = 0.06 for the highest vs. lowest tertile of folate intake). In contrast, it was positively associated with APC⁺ colon tumors in men (highest vs. lowest tertile: RR 2.77, 95% CI 1.29–5.95, P_trend = 0.008) and was even stronger when the lack of hMLH1 expression and K-ras mutations were excluded (RR 3.99, 95% CI 1.43–11.14, P_trend = 0.007). Such positive associations were not observed among women; nor was folate intake associated with rectal cancer when APC mutation status was taken into account. Relatively high folate consumption reduced the risk of APC^– colon tumors, but folate intake was positively associated with APC⁺ colon tumors among men. These opposite results may indicate that folate enhances colorectal carcinogenesis through a distinct APC mutated pathway.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

A well-characterized molecular alteration in colorectal carcinogenesis is the occurrence of mutations in the adenomatous polyposis coli (APC)⁷ gene. Somatic mutations in this tumor suppressor gene were found mainly in its mutation cluster region in a significant proportion of sporadic colorectal carcinomas, varying from 34 to >80% (5–8). This underscores the importance of APC gene mutations in sporadic colorectal carcinogenesis. In an earlier study in colorectal cancer patients (8) we found that 37% of the patients had tumors harboring an APC mutation resulting in a stop codon, which leads to a truncated and therefore inactive APC protein. In addition, Diergaarde et al. (9) found an indication for a positive association between folate intake and colorectal adenomas, but the associations did not differ between adenomas with or without truncating APC mutations. In another study, Diergaarde et al. (10) observed a differential association between the consumption of green leafy vegetables, an important source of dietary folate, and the risk of carcinomas with or without truncating APC mutations.

The exact mechanism through which folate may alter APC functionality remains unclear, however. Possibly, there is an indirect effect of decreased activity of the DNA repair gene O⁶-methylguanine DNA methyltransferase (O⁶-MGMT), which was found to be associated with hypermethylation of its promoter region (11). Consequently, this can lead to an increased risk of tumors with G:C > A:T mutations in other key genes, like APC, involved in colorectal cancer (12,13).

Several studies have suggested that colorectal tumors may arise through distinct pathways based on specific molecular alterations. In this respect, mutations in APC and Kirsten ras (K-ras) occur less often in tumors with microsatellite instability (MSI) compared with microsatellite stable ones (14–17). MSI is known to be strongly associated with the lack of expression of the DNA mismatch repair gene human mut-L homologue 1 (hMLH1) (18,19). Tumors harboring this type of instability occur predominantly in the proximal colon, are often poorly differentiated, and are more frequently present in women at an older age (14,20). Furthermore, exogenous factors such as smoking behavior (21,22) and dietary factors (7,23–25) may be differentially associated with the risk of cancer development in MSI and non-MSI tumors. In addition, differential associations were also observed between folate intake and mutations in K-ras (26–29). Therefore, it is important to account for potential aberrations in K-ras and hMLH1 and for differences in tumor location and sex when assessing the potential effect of dietary folate on colorectal cancer risk and APC mutations.

In view of these findings, this study aimed to investigate the associations between folate intake and the risk of colon and rectal cancer, with and without truncating APC mutations. We also assessed whether these associations were independent of K-ras mutations and hMLH1 expression. The study was conducted within the framework of the Netherlands Cohort Study on diet and cancer (NLCS), of which the study population consumed relatively low levels of dietary folate (insofar as folic acid fortification is not allowed in the Netherlands) compared with some other Western countries.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Study population. The participants in this study were incident colon and rectal cancer patients and subcohort members from the NLCS, which has been described in detail elsewhere (30). Briefly, the study was initiated in 1986 and includes 58,279 men and 62,573 women, aged 55–69 y at baseline, who originated from 204 Dutch municipalities with computerized population registers. Numbers of subcohort members and cases are displayed in a flow chart (Fig. 1). At baseline, participants completed a self-administered food frequency questionnaire that also contained questions about other risk factors for cancer. The entire cohort has been monitored for cancer occurrence by annual record linkage to the Netherlands Cancer Registry (9 cancer registries in The Netherlands) and to Pathologisch Anatomisch Landelijk Geautomatiseerd Archief (PALGA), a nationwide network and registry of histopathology and cytopathology reports (31). Accumulation of person-time in the cohort was estimated through biennial vital status follow-up of a subcohort of 5000 men and women who were randomly selected after baseline exposure measurement. Cases with prevalent cancer other than nonmelanoma skin cancer were excluded from this subcohort, which left 4774 men and women eligible for analysis.

View larger version (15K): [in this window] [in a new window]   Figure 1  Flow diagram of subjects on whom the analyses were based 1 Netherlands Cancer Registry. 2 Pathologisch Anatomisch Landelijk Geautomatiseerd Archief. 3 Patients with rectosigmoid tumors were not included in the analyses. 4 Adenomatous polyposis coli. 5 Mutation cluster region. 6 Kirsten ras. 7 human mut-L homologue 1.

    Tissue samples. Tumor material of the colorectal cancer patients was collected after approval by the ethical review boards of Maastricht University, the NCR, and PALGA (32). In addition, all pathology laboratories in The Netherlands agreed to make relevant tissue samples available from PALGA upon request. From 815 tissue samples that were scattered over 54 pathology laboratories in the Netherlands, 734 samples (90%) could be traced and were retrieved between August 1999 and December 2001.

    Gene mutation analyses. Genomic DNA was isolated from the paraffin sections after macrodissection of tumor cells as previously described (32). Gene mutation analyses of the mutation cluster region in APC (codons 1286–1520), was performed as previously described (8). Briefly, nested PCR was used to amplify the mutation cluster region in 4 overlapping DNA fragments, and the purified fragments were sequenced. Two observers independently evaluated the sequence patterns and data entry. For 72 colorectal cancer patients, one or more fragments of the APC gene could not be analyzed completely, leaving 662 patients for data analysis.

The exon 1 fragment of the K-ras oncogene, spanning codons 8–29, was analyzed successfully for all 734 patients using nested PCR, followed by direct sequencing of purified fragments as described (32).

    hMLH1 expression analysis. Immunohistochemical analyses were performed and scored on 4-µm sections of formalin-fixed, paraffin-embedded cancer tissue and adjacent normal tissue using a monoclonal antibody against hMLH1, as previously described (7). Two investigators reviewed the immunohistochemical staining profiles independently and discrepancies were reexamined and discussed with a pathologist until consensus was reached. hMLH1 expression status was determined successfully in 721 (98%) of 734 patients.

    Food frequency questionnaire. The self-administered questionnaire was a 150-item semiquantitative food frequency questionnaire, which concentrated on habitual consumption of food and beverages during the year preceding the start of the study and also contained questions about body weight and length, smoking status, physical activity, and family history of colorectal cancer. Daily mean nutrient intakes were calculated as the cumulated product of the frequencies and portion sizes of all food items and their tabulated nutrient contents from the Dutch Food Composition Table (NEVO table, 1986) (33). The questionnaire was validated through comparison with a 9-d diet record (34). Questionnaire data were key-entered twice for all incident cases in the cohort and for all subcohort members in a blinded manner with respect to case or subcohort status. This was done to minimize observer bias in coding and interpretation of the data.

Folate data were derived from a validated liquid chromatography trienzyme method (35) used to analyze the 125 most important Dutch foods contributing to folate intake (36). Mean daily intakes of all other relevant nutrients were calculated using the computerized Dutch Food Composition Table (33). Dietary supplement data were also obtained via the food frequency questionnaire. However, the use of B-vitamins and/or multivitamin supplements was low (7 and 4%, respectively), and folic acid was generally not included in supplements in The Netherlands in the late 1980s. Therefore, folic acid supplement use probably plays a very minor role in our study population, and dietary supplement use was not further accounted for in the analyses. Cases and subcohort members with incomplete or inconsistent dietary data were excluded from analyses (27,34). Hence, 446 colon cancer cases, 160 rectal cancer cases, 76 rectosigmoid cancer cases, and 4343 subcohort members remained.

    Statistical analyses. Data analyses were conducted overall, as well as stratified for men and women, and for colon and rectal tumors with and without truncating APC mutations, described here as APC⁺ tumors and APC^– tumors, respectively. The group of cases without truncating APC mutations consisted of cases with tumors containing a wild-type APC gene (28%), a missense mutation (29%), or a silent mutation (6%). Because the number of patients with a rectosigmoid tumor was too small for adequate stratified analyses, we included these patients only when assessing associations for all colorectal tumors combined. Furthermore, the rectosigmoid is regarded as a clinically applied term rather than an anatomically defined transitional zone between the colon and rectum (32).

The analyses were repeated for tumors with APC mutations, excluding those tumors that also contained activating K-ras mutations and were hMLH1 deficient. Of 125 truncating APC colon tumors, 64 (49%) exclusively contained a truncating APC mutation. To investigate the effect of folate on specific point mutations, analyses were conducted for tumors with G:C > A:T mutations in APC irrespective of the presence of truncating APC mutations or other gene defects.

The intake of dietary folate and other baseline characteristics were evaluated for subcohort members and colon and rectal cancer cases, with or without APC mutations, by calculating the mean and SD of the continuous variables, which included folate intake (µg/d), age (y), BMI (kg/m²), energy (kJ/d), alcohol (g/d), total fat (g/d), fiber (g/d), vitamin C (mg/d), riboflavin (mg/d), vitamin B-6 (mg/d), iron (mg/d) and methionine (mg/d). Distributions of the variables for family history of colorectal cancer (yes/no), smoking status (never/ex/current smoker), and physical activity during leisure time (<30, 30–60, 60–90, >90 min/d) were also calculated. Differences in mean values or distributions of variables between groups of cases with and without truncating APC mutations were tested with the Student's t test, the Mann-Whitney U test, or the chi-square test where appropriate.

Cox proportional hazards regression models were used to estimate age-adjusted and multivariate-adjusted gender-specific incidence rate ratios (RR) and corresponding 95% CI of colon and rectal cancer for tertiles of folate intake. In addition, associations were estimated for tumors with or without truncating APC mutations. Standard errors of the RRs were estimated using the robust Huber-White sandwich estimator to account for additional variance introduced by sampling from the cohort (37). The proportional hazards assumption was tested using the scaled Schoenfeld residuals (38). Tests for dose response trends over the different tertiles of folate intake were estimated by fitting the ordinal exposure variables as continuous variables and evaluated using the Wald test.

The covariates included in the multivariate analyses were those found to significantly contribute to the multivariate model (P 0.05) for colon and/or rectal cancer, or to influence the RR by >10%. This applied to the variables for age, family history of colorectal cancer, BMI, energy, fiber, vitamin C, riboflavin, vitamin B-6, iron and methionine. After excluding subjects with missing information on 1 of these covariates, 4200 subcohort members remained for statistical analyses, as well as 399 colon and 129 rectal cancer cases with complete APC and K-ras mutation analyses, and 394 colon and 128 rectal cancer cases with additional complete hMLH1 expression analyses.

Several factors were previously found to be associated with colorectal cancer risk or to be capable of modifying the association between dietary folate and colorectal cancer risk, i.e., smoking (22,39,40), alcohol (41,42), riboflavin, B-6 (43,44), and iron intake (45). We therefore determined possible effect modification of these factors, as well as for sex, by testing for interaction and by stratified analyses.

All statistical analyses were performed with the STATA statistical software package (Intercooled STATA, version 9.1).

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
We explored mean dietary intakes and other characteristics measured at baseline for cancer cases and subcohort members. Folate intake among males with APC⁺ colon tumors was higher compared with males with APC^– colon tumors (Table 1). Furthermore, we observed that intakes of riboflavin and iron were significantly higher among men with APC⁺ colon tumors compared with men with APC^– colon tumors. All other characteristics presented in Table 1 did not differ substantially between tumors with and without truncating APC mutations.

Regarding rectal cancer, we observed that intake of dietary folate was neither associated with overall cancer risk nor with rectal cancer, with or without APC truncating mutations, in either men or women (see Table 3). However, when conducting the analyses for APC⁺ tumors without a K-ras mutation and with hMHL1 expression, folate tended to be positively associated with rectal cancer risk among men (RR 3.69, CI 0.97–13.98; P_trend = 0.06).

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
In this study, we investigated the associations between folate intake and colorectal carcinoma risk with and without truncating APC mutations. We observed that folate intake in the 2nd and highest tertile reduced the risk of APC^–colon tumors among men, whereas folate intake was positively associated with APC⁺ colon tumors in men. This positive association among men was even stronger for APC⁺ colon tumors, and appeared in rectal tumors, when tumors with additional aberrations in K-ras and with hMLH1 expression were excluded.

To our knowledge, the relation between folate and APC mutations was investigated only once before in colorectal adenomas by Diergaarde et al. (9), but, in contrast to our study, no differences were observed between the APC^– and APC⁺ endpoints. Reasons for this discrepancy might be that adenomas were studied instead of carcinomas, and that a case-control design was used with selected cases vs. a case-cohort design with incident cases in our study. Furthermore, in the study of Diergaarde et al. (9), the analyses were not stratified for gender, which may have attenuated potential associations in men. On the other hand, the ranges of dietary folate intake within the tertiles of intake were comparable to those in our study population. In another study by Diergaarde et al. (10), intake of green leafy vegetables, an important source of dietary folate, was inversely associated with APC^–colon carcinomas. Although the association with folate intake was not calculated separately in that study, the results suggest a similarity with the inverse association between folate and APC^– colon carcinomas in our study. However, the reason why the protective influence of folate is confined to these tumors remains unclear and needs further investigation.

After determining potential interactions, we found that sex significantly modified the effect of folate intake on overall colon cancer risk, as well as on APC⁺ colon tumors. For this reason, we presented the data for men and women separately despite the drawbacks of thereby creating smaller subgroups and less-precise estimations. To minimize the potential danger of reporting chance findings, however, we also conducted analyses for all colorectal tumors combined, with and without stratifying for sex. We then observed that folate intake was again positively associated with APC⁺ colorectal tumors without a K-ras mutation and with hMHL1 expression, and that this effect was only present among men.

Although one could argue that the number of cases in some subgroups should preferably have been higher, we emphasize that the selection of patients in this study was based on the availability of tissue samples with sufficient extracted DNA as well as the completeness of analyses of the K-ras and APC genes and hMLH1 expression. This led to a reduction of the number of cases that could be included in the analyses. However, a tissue sample, as well as a sufficient amount of DNA, was available for 90% of the patients. Moreover, the overlap of available molecular analyses was high (89%). Finally, it is important to realize that the characteristics of these patients with regard to age, sex, family history of colorectal cancer, smoking behavior, and dietary factors differed neither from the 815 patients initially identified nor from the 734 patients with sufficient tumor DNA. It is therefore unlikely that bias occurred due to the selection of patients.

High folate intake resulted in an increased risk of APC⁺ colon tumors among men. However, this was not expected, given the hypothesis that folate prevents DNA damage. Because it is reasonable to assume that aberrations in other key genes in colorectal carcinogenesis may also be associated with folate intake, we included K-ras mutation status and hMLH1 expression in our analyses to exclude their potential underlying influence. The observations that APC and K-ras mutations may be inversely associated with MSI (14–17) and with hMLH1 expression (18,19) are important reasons to do so. APC mutated colon tumors, without additional K-ras mutation and with hMLH1 expression, were even more positively associated with folate intake in men, and the positive association also appeared for this type of rectal tumor among men. Interestingly, folic acid supplementation might have a cancer-promoting effect in people with already-existing, undiagnosed premalignant or malignant lesions (4,46). APC mutations play an important role in both tumor initiation as well as in later stages of colorectal cancer development (47). The development of lesions containing early APC mutations is possibly more sensitive to a high folate intake; that is, the protective influence of dietary folate on cancer progression might be decreased after an APC mutation has occurred compared with tumors that were initiated otherwise.

Other recent studies confirm that multiple alternative genetic pathways to colorectal cancer may exist, because it was found that only a small number of colorectal tumors (5– 6%) harbored mutations in all 3 key genes K-ras, APC, and TP53 (48,49). We observed strong positive associations for tumors containing exclusively APC mutations in men only. This may indicate that folate potentially mediates an APC mutated pathway in the colon and rectum in men, but that its effect differs between men and women. However, the reasons for these differences are unknown, and clearly, other studies are warranted to confirm or reject this.

The relative risks of tumors harboring G:C > A:T point mutations in APC were slightly decreased among women with colon tumors. Decreased activity of the DNA repair gene O⁶-MGMT by epigenetic hypermethylation might be an indirect mechanism through which folate intake modifies colorectal cancer risk (11). The O⁶-MGMT protein removes adducts from the O⁶ position of guanine in DNA (12), which in turn prevents G:C > A:T point mutations. Previously, we reported that O⁶-MGMT promoter hypermethylation may partly be due to a low folate status (50). It has also been reported that that O⁶-MGMT hypermethylation is associated with G:C > A:T mutations in K-ras and TP53 (12,13,27). However, others did not observe an association between O⁶-MGMT expression and G:C > A:T mutations in the APC gene alone (51). Further research is therefore needed to investigate whether APC mutations might occur via decreased O⁶-MGMT functionality.

A potential protective effect of folate intake on colorectal cancer risk has been demonstrated only in 10 of 23 epidemiological studies investigating this association (2). In addition, we observed higher risks of colorectal cancer in some subgroups even though intakes of dietary folate in our study population were relatively low compared with some other Western countries. This raises the question as to whether high folate intake should be a reason for concern in terms of colorectal cancer risk, particularly in view of differences in recommended dietary intakes between countries and the mandatory fortification of cereals with folic acid in the United States and Canada. It is therefore of crucial importance to gain more insight into the role that dietary folate plays in colorectal carcinogenesis with genetic aberrations like truncating APC mutations in future studies.

In conclusion, the results of this study indicate that an inverse association between relatively high folate consumption and carcinogenesis is limited to APC^– colon tumors, especially in men. Combined with the higher relative risks of APC⁺ colon and rectal tumors without additional aberrations in 2 other key genes involved in colorectal cancer in men, these findings may indicate that folate intake exerts an effect on specific genetic pathways, i.e., an APC mutated pathway. Tumors that arise through distinct genetic pathways on the basis of specific genetic aberrations, such as truncating APC mutations, possibly have a unique etiology, and folate may have a different effect on these pathways in men and women.

	ACKNOWLEDGMENTS

 
We thank Dr. M. Brink for the collection of tissue samples; Dr. M. Brink, Dr. M. Lüchtenborg, P. Wark, G. Roemen, and K. Wouters for molecular analyses; Dr. L. Schouten, S. van de Crommert, H. Brants, J. Nelissen, C. de Zwart, M. Moll, W. van Dijk, M. Jansen, and A. Pisters for data management; and H. van Montfort, T. van Moergastel, L. van den Bosch, and R. Schmeitz for programming assistance. Finally, we would like to thank Dr. A. Volovics and Dr. A. Kester for statistical advice.

	FOOTNOTES

 
¹ Financial support was granted by the Dutch Cancer Society (UM2004-3171 and UM 99-1980) and The Netherlands Organization for Scientific Research (980-10-26).

⁶ Present address: Cancer Research Center of Hawaii, University of Hawaii, Honolulu.

⁷ Abbreviations used: APC, adenomatous polyposis coli; hMLH1, human mut-L homologue 1; K-ras, Kirsten ras; MSI, microsatellite instability; NLCS, Netherlands Cohort Study; O⁶-MGMT, O⁶-methylguanine DNA methyltransferase; PALGA, Pathologisch Anatomisch Landelijk Geautomatiseerd Archief; RR, incidence rate ratio.

Manuscript received 5 July 2006. Initial review completed 31 July 2006. Revision accepted 23 September 2006.

	LITERATURE CITED

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 

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