1 Department of Epidemiology and Biostatistics, University of South Carolina, Arnold School of Public Health, Columbia, SC, USA, 2 South Carolina Cancer Center, Columbia, SC, USA, 3 Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, USA, 4 Department of Pathology, University of South Carolina School of Medicine, Columbia, SC, USA and 5 Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA
6 To whom correspondence should addressed at: University of South Carolina, 14 Richland Medical Park, Suite 500, Columbia, SC 29203, USA. Tel: +1 803 434 3707; Fax: +1 803 434 3795; Email: dawen.xie{at}palmettohealth.org
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Abstract |
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Abbreviations: BMI, body mass index [weight (kg)/height (m)2]; CI, confidence interval; NSAID, non-steroidal anti-inflammatory drug; OR, odds ratio; PPAR, peroxisome proliferator-activated receptor
; WHR, waist:hip ratio
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Introduction |
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Sporadic cancer, with no apparent familial or inherited predisposition, accounts for 70% of colorectal cancer in the total population. Sporadic colon cancer risk is increased in persons >50 years of age, probably as a result of dietary and other environmental factors. Colorectal cancer develops as the result of a progressive accumulation of genetic and epigenetic alterations that lead to the transformation of normal colonic epithelium to colon adenocarcinoma (5). While environmental factors clearly play an important role in colorectal cancer etiology, genetic factors also appear to be very important in developing colorectal cancer. Additionally, geneenvironment interactions in the pathogenesis of colorectal cancer are plausible and under investigation. In summary, colorectal cancer appears to be related causally to genes, the environment and geneenvironment interactions, although the risk attributable to each is not clear.
Peroxisome proliferator-activated receptor (PPAR
) is a member of the nuclear hormone receptor superfamily that plays an important role in body plan specification, cell differentiation and regulation of metabolism (6,7). PPAR
, as a heterodimer with the retinoid X receptor
, binds DNA and regulates target gene expression (8). PPAR
regulates the expression of numerous genes (e.g. aP2, PEPCK, TNF
and UCP-1) involved in lipid metabolism, cellular differentiation, glucose homeostasis, eicosanoid signaling and inflammation (912). Specifically, PPAR
has an important role in mediating adipocyte differentiation and fat metabolism regulation (13). PPAR
is expressed at the highest levels in adipose tissue, but high levels also are found in other epithelial tissues, including colon, prostate and breast (1416). Both colonic epithelial cells and colon cancer cells have been shown to highly express PPAR
(17,18). PPAR
has been implicated as playing a role in colon cancer progression by controlling cell growth and differentiation. Experimental studies have shown that ligand activation of PPAR
inhibits cell growth, induces apoptosis and promotes differentiation in colon cell lines (1921). That PPAR
is activated by fatty acids and other arachidonic acid metabolites suggests links between fatty acid metabolism, control of gene transcription and risk of colon cancer. Tumor suppressor properties and certain inactivating mutations in the PPAR
gene have been found in human colon cancer (22). In addition, in animal models application of PPAR
ligands was found to reduce chemically induced colonic inflammation and aberrant crypt focus formation (23). The study of subjects harboring natural mutations or polymorphisms within PPAR
provides compelling genetic evidence for it having roles in glucose homeostasis, lipid metabolism and fat mass determination (24).
A common structural polymorphism in the PPAR gene, CCA
GCA, producing a Pro
Ala substitution at codon 12 (Pro12Ala), has been detected. The Pro12Ala polymorphism has been associated with altered lipid profiles, lower fasting insulin concentrations, improved insulin sensitivity and a reduced risk of type II diabetes and the metabolic syndrome (2527). This amino acid is located in the PPAR
domain that enhances ligand-independent activation (28). The Pro
Ala change may cause a conformational change in the protein, thus affecting its activity.
These findings suggest that the Pro12Ala polymorphism could plausibly play a role in the etiology of colorectal adenomas, a precursor lesion of colorectal cancer. No known published epidemiological studies have evaluated the Pro12Ala polymorphism of PPAR and risk for incident sporadic colorectal adenomatous polyps and no studies have investigated potential interactions between dietary and other environmental risk factors and Pro12Ala genotypes and risk for colorectal neoplasms.
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Materials and methods |
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Cases were defined as patients with at least one adenomatous polyp and controls were identified as those patients without adenomatous polyps. Polyps were removed at colonoscopy and examined by a study pathologist using criteria from the National Polyp Study (30). Information on polyp location, number, size, shape, histological type and degree of dysplasia were collected.
Study participants were sent self-administered questionnaires before their colonoscopy visit. Information on smoking habits, alcohol intake, non-steroidal anti-inflammatory drug (NSAID) use, physical activity, medical history, anthropometry (height, weight and waist and hip circumferences), family history of polyps or colorectal cancer, reproductive history and reasons for colonoscopy were collected using the mailed questionnaires, which were collected from each subject on the colonoscopy visit. Dietary information was assessed using an adaptation of the Willett semi-quantitative food frequency questionnaire (153 items), which was expanded to include additional vegetables, fruit and low fat foods (31). Physical activity information was obtained using a modified Paffenbarger questionnaire (32). Alcohol intake data were collected using alcohol consumption questions in both the food frequency questionnaire and the personal history portion of the questionnaire. Blood was drawn and stored at 70°C for possible later measurement of various genotypes.
Among all four clinical sites, 669 participants were found to be eligible for the study initially. Of these, 617 were contacted and 417 participants (62.3% of total eligible participants) signed the consent form and participated in the study. Of the 417 participants, 259 had some type of polyp and of these 179 had adenomatous polyps. Nine of the 417 total participants were subsequently determined to be ineligible for the study and an additional 8 participants had incident colon cancer and were not eligible for the primary casecontrol analyses, thus, 400 possible participants were available for genotypic analysis. There was sufficient DNA from 375 of these 400 participants to enable PPAR polymorphism genotyping.
Laboratory methods
Genomic DNA was obtained from stored white blood cells digested in 500 µl of lysis buffer (50 mM TrisHCl, pH 8.5, 1 mM EDTA, 0.2% SDS, 200 g/ml proteinase K) overnight at 55°C with shaking. The digestate was precipitated directly with isopropanol and the pellets were washed with 70% ethanol. The genomic DNA pellets (50100 µg) were dissolved in 300800 µl of TE buffer, of which 1 µl was used for each PCR reaction.
The PPAR Pro12Ala polymorphism was detected by the PCRrestriction fragment length polymorphism method. We designed two primers to amplify a 257 bp fragment of the PPAR
gene following the published sequence. The PCR primers were: (forward) 5'-CCAATTCAAGCCCAGTCCTTTC-3' and (reverse) 5'-GCAGACAGTGTATCAGTGAAGGAATCGCTTTCCG-3'. The PCR reactions were performed on a Perkin Elmer GeneAmp System 9700 according to the manufacturer's protocol. Specifically, these reactions were carried out in 50 µl of 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.0 mM MgCl2, 0.2 mM dNTP, 1 U Taq polymerase (Gibco-Invitrogen) and 0.4 µM each oligonucleotide primer. The reactions were heated to 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 52°C for 30 s and 72°C for 45 s. At the end, the reactions were extended for 7 min at 72°C. After PCR amplification, each PCR product was subjected to BstUI digestion prior to electrophoresis. The DNA fragments were separated using 3% 2:1 Nusiev/SeaKem agarose gel. The genotypes were determined as follows: a single 257 bp fragment for the CC (Ala12Ala) genotype, two fragments of 223 and 34 bp for the GG (Pro12Pro) genotype and three fragments of 257, 223 and 34 bp for the CG (Pro12Ala) genotype.
Statistical data analysis
All statistical analyses were conducted using SAS Software version 8.2e from the SAS Institute (Cary, NC). Baseline characteristics of cases and controls were compared using analysis of covariance for continuous variables and 2 tests for categorical variables. Allelic frequencies for polymorphic PPAR
C/G alleles were compared with those in previous study populations. PPAR
genotype (CC, CG and GG) distributions for cases and controls were tested for adherence to the HardyWeinberg equilibrium. The odds ratio (OR) and 95% confidence interval (CI) were calculated to measure the association between exposure and disease (i.e. presence of adenomatous polyps).
Multiple logistic regression models, using Proc LOGISTIC in SAS, were fitted to estimate the risk of colorectal adenoma risk in relation to the genetic polymorphisms of PPAR while controlling for potential confounders. Several risk factors were examined as possible confounders or effect modifiers of the PPAR
Pro12Ala genotypecolorectal adenoma association. Among these were age, sex, race, body mass index (BMI), family history of colorectal cancer in the first degree, smoking, alcohol consumption, NSAID use and total dietary intakes of fat, energy, calcium, meat, vegetables and fruit and various antioxidant micronutrients. Criteria for inclusion of any covariate in the final model included: (i) biological plausibility; (ii) whether it fitted the model at P = 0.1; (iii) whether it altered the OR for the primary exposure variable, PPAR
Pro12Ala genotype, by
10%. Final models for genotype effects included age, sex, family history of colorectal cancer in the first degree, ever smoked status and current use of NSAIDs. Models involving assessing possible interactions between genotypes and various key risk factors included the same covariates plus total energy intake. Age and total energy intake were fitted as continuous variables in the main model.
Polytomous logistic regression models were used to analyze the association between PPAR genotypes and colorectal adenoma according to adenoma characteristics. The polytomous logistic regression model is a kind of logistic regression model that can be used when the dependent variable has more than two levels, instead of the usual dichotomy typically employed in logistic regression. Testing of interactions was based on the joint effects approach, which involves comparison of the joint observed with expected odds ratios. Models for examining joint effects between selected effect modifiers and PPAR
genotypes (e.g. joint effect of smoking and genotype) were evaluated and included the other primary exposure variables as covariates. Selection of potential effect modifiers (possible interaction variables) was based on previous literature, biological plausibility and whether or not risk estimates differed substantially across strata. In models that examined the joint effect of age with genotype, age was categorized into a dichotomous variable (
57 years or >57 years) based on the median value among controls. Specific nutrient intakes were dichotomized into low or high based on the sex-specific medians from the distributions of intakes among controls, while ever smoked, current NSAID use, family history of colorectal cancer in the first degree and ever consumed alcohol were categorized as dichotomous variables (yes/no).
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Results |
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The distribution of PPAR genotypes, age- and sex-adjusted and multivariate-adjusted ORs with corresponding 95% CI are shown in Table II. The frequency of the GG (Pro/Pro) genotype was 79.1% in cases and 72.2% in controls. Similarly, the frequency of having at least one C allele (GC, Pro/Ala and CC, Ala/Ala) was 20.9% among cases and 27.8% among controls. These genotype frequencies were consistent with those in a previous study (35). Because the proportion of subjects who were homozygous was small (
3%), we combined the heterozygous and homozygous variant genotypes to represent the variant genotype for the risk analysis. When adjusted only for age and sex, the OR for those with the GC and CC genotypes compared with those with the GG genotype was 0.71 (95% CI 0.431.17). The multivariate-adjusted OR was 0.65 (95% CI 0.391.09).
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Discussion |
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Several biologically plausible mechanisms support a possible protective effect of the PPAR Ala12 polymorphism on colorectal adenoma. First, PPAR
is an important regulator of lipid and lipoprotein metabolism and glucose homeostasis. Previous studies have found that PPAR
Ala12 is associated with higher insulin sensitivity, which could result in a reduced release of free plasma fatty acids by adipocytes (36). Insulin stimulates pathways that increase levels of insulin-like growth factor and both insulin and insulin-like growth factor promote mitosis and cell proliferation and inhibit apoptosis in normal and cancer cells of the colonic epithelium (3739). Recent epidemiological evidence also supports the hypothesis that high insulin exposure or insulin resistance may be associated with colorectal adenoma and cancer risk (40,41). Thus, a potential protective effect of the PPAR
C allele on colorectal adenoma risk may be related, either directly or indirectly, to increased insulin sensitivity, improvement of insulin resistance or both. Second, prostaglandin products, generated by cyclooxygenase (COX1/COX2), have long been implicated in colon tumorigenesis and PPAR
may simply function as a downstream mediator that results in reduced biosynthesis of these products (42). Finally, activation of PPAR
by artificial or natural ligands was found to inhibit the NF-
B and STAT3 inflammation pathways, thereby inducing growth inhibition associated with a delay in the G1 phase of the cell cycle and to increase differentiation and apoptosis in cultured human colorectal cells (43,44).
Given that a potential protective effect of this polymorphism may act in concert with other environmental risk factors, we explored interactions between environmental factors and PPAR genotypes. In this analysis there were no statistically significant interactions between this polymorphism and plausible risk factors. However, smoking was statistically significantly associated with an increased risk for adenoma among those with the homozygous wild-type genotype (GG), while there was no apparent increase in risk among those with at least one C allele. Moreover, inverse associations of adenoma with sex (female), alcohol, NSAID use, BMI and WHR tended to be stronger among those who also carried at least one variant allele. Although the Pro12Ala polymorphism was associated with only a marginally significant (possibly due to the relatively small sample size) inverse association with colorectal adenoma risk, these results are consistent with a protective effect by this polymorphism, and the data suggest that the polymorphism may confer a lower risk of colorectal adenoma by interacting with other environmental factors.
PPAR is an important regulator of lipid metabolism and dietary fatty acids, which can act as PPAR
ligands that may regulate gene expression to induce differentiation and apoptosis. Thus, an interaction between dietary fat and this polymorphism was investigated in this study. We found no evidence for such an interaction. On the other hand, no association was detected between dietary fat intake and colorectal adenoma risk. Evidence from experimental and epidemiological studies suggests that dietary calcium and vitamin D may modulate and inhibit colorectal carcinogenesis (4547). Recent findings indicate that high calcium and vitamin D diets can reduce adipocyte fatty acid synthesis and thereby modulate energy metabolism and attenuate obesity (4850). Interactions between calcium and vitamin D intake and this lipid metabolism gene polymorphism were investigated in our study. Similar to the findings for dietary fat, no such interactions were observed in this study. However, risk for adenoma tended to be lower among variant allele carriers with high calcium intakes. The explanation for these findings might in part be due to use of the Willett Food Frequency Questionnaire, a self-report dietary assessment method. This self-reported measurement method has been shown to result in non-differential misclassification, which can bias estimates of the association between fat intake and adenoma risk towards the null value, as well as reduce our ability to observe an interaction between fat intake and the polymorphism (51). Other evidence exists that this type of instrument is also subject to a variety of biases that further complicate interpretation of questionnaire-derived data (5255).
We also investigated the association of the polymorphism with colorectal adenoma risk according to characteristics of the adenoma polyps. There were no significant associations between this polymorphism and different characteristics of adenomatous polyps. The small sample size may account for these non-significant results. On the other hand, consistent inverse associations of this polymorphism with adenoma risk regardless of characteristics were observed. Furthermore, although not statistically significant, the inverse associations appeared to be stronger for adenomas with more advanced characteristics, suggesting that the polymorphism may play a stronger role in adenoma progression than initiation. It is necessary to evaluate these relationships in a larger population study.
Most studies of PPAR have focused on the association between this polymorphism and type II diabetes. This polymorphism has been investigated extensively for associations with obesity and type II diabetes mellitus, and the Ala allele is associated with a modest inverse association with type II diabetes (OR 0.72, 95% CI 0.520.99) (56). The PPAR
Ala allele was found to have decreased transcriptional activity compared with the Pro isoform (57). Although there have been several studies of PPAR
function and colon cancer in basic science studies, there have been only two studies reported in humans. Two published casecontrol studies investigated this polymorphism with respect to colorectal cancer risk. One study, including 39 Japanese cancer patients, reported that the Pro12Ala polymorphism may be associated with colorectal cancer among those whose K-ras gene is not mutated (58). The other study, a hospital-based casecontrol study conducted in Spain, indicated a significant inverse association of the PPAR
Ala12 polymorphism with colorectal cancer risk (35). The OR was 0.56, which was very close to the point estimate obtained in our adenoma study. That study included a larger sample size (cases 377, controls 326) than ours. Colorectal adenomas appear to share the spectrum of risk factors seen with colon cancer, although for adenomas tobacco smoking is a clearer and more consistent risk factor.
This study has several strengths. To our knowledge there are no other studies that investigated an association of the PPAR Pro12Ala polymorphism with colorectal adenoma risk. Data were carefully collected from this community-based, colonoscopy-based casecontrol study on adenoma characteristics, which allowed us to assess PPAR
genotype and adenoma risk according to different characteristics of the adenomas. In addition, self-administered questionnaire data were collected prior to diagnosis in order to reduce recall bias. Because of the high prevalence of adenoma in the general population, cases and controls were accurately defined by full colonoscopic examination to minimize bias from misclassification of adenoma status.
This study also has several limitations. First, although the colonoscopy-based design minimized possible misclassification bias, the study population may not have been fully representative of the general population because such subjects may be at higher risk for colorectal neoplasms. For example, patients receiving colonoscopies who were classified as controls may be at higher risk than the general population of later developing polyps and therefore have risk profiles more similar to those of the cases. However, this would tend to attenuate associations, therefore, our findings may actually underestimate the influence of the PPAR Ala polymorphism on risk for colorectal adenoma. Second, our sample size was small, which limited statistical power to detect true associations, especially for interactions. However, the findings were generally consistent with both a priori hypotheses and findings from two previous studies of colorectal cancer, and thus warrant further study. Moreover, we investigated the association of this polymorphism with adenoma rather than cancer risk. Colorectal adenomas appear to share the same spectrum of risk factors seen with colon cancer. However, adenomas are precursors for colorectal cancer and not all adenomas progress to cancer. Further research is needed to determine the association between this polymorphism and colorectal cancer. On the other hand, an advantage of studying adenomas is that they are asymptomatic, which limits the possibility of recall bias to some extent. Study of adenomas may help identify factors that are important in the etiology of colorectal cancers.
In conclusion, our results, despite lacking statistical significance at < 0.05 in many instances, were consistent with the a priori hypothesis and thus suggested that the Ala12 variant of PPAR
may favorably modulate susceptibility to colorectal adenoma risk. The estimated associations of the polymorphism with adenoma risk tended to differ depending on other environmental factors, which suggests potential geneenvironment interactions. Understanding how specific modulation of PPAR
influences metabolism in humans may result in more targeted dietary recommendations and accelerate the development of novel pharmacological agents useful for preventing colorectal adenoma and cancer. Larger studies with better measurements of dietary covariates are needed to answer the intriguing questions raised by this study.
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Acknowledgments |
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References |
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