Arachidonate lipoxygenase (ALOX) and cyclooxygenase (COX) polymorphisms and colon cancer risk

Julie E. Goodman1,2, Elise D. Bowman1, Stephen J. Chanock3, Anthony J. Alberg4 and Curtis C. Harris1,5

1 Laboratory of Human Carcinogensis, 2 Cancer Prevention Fellowship Program, Division of Cancer Prevention and 3 Advanced Technology Center, National Cancer Institute, Bethesda, MD 20892, USA and 4 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA

5 To whom correspondence should be addressed Email: curtis_harris{at}nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the human colon, arachidonic acid is metabolized primarily by cyclooxygenase (COX) and arachidonate lipoxygenase (ALOX) to bioactive lipids, which are implicated in colon cancer risk. Several polymorphisms in ALOX and COX genes have been identified, including G-1752A, G-1699A and Glu254Lys in ALOX5; Gln261Arg in ALOX12; Leu237Met and Val481Ile in COX1; and C-645T and Val511Ala in COX2. Because of the significant role of arachidonic acid metabolism in colon cancer, we hypothesized that these polymorphisms could influence susceptibility to colon cancer. We addressed this hypothesis in African-Americans and Caucasians using colon cancer cases (n = 293) and hospital- (n = 229) and population-based (n = 304) control groups. Polymorphisms did not differ between the control groups (P > 0.05); thus, they are combined for all analyses presented. ALOX5 Glu254Lys and COX2 C-645T and Val511Ala allele frequencies differed between Caucasians and African-American controls (P < 0.001). The ALOX5 –1752 and –1699 polymorphisms were in linkage disequilibrium (P < 0.001) and associated with a decreased risk in Caucasians in ALOX5 haplotype analyses (P = 0.03). Furthermore, an inverse association was observed between A alleles at positions –1752 and –1699 of ALOX5 and colon cancer risk in Caucasians, but not in African-Americans. Caucasians with A alleles at ALOX5 –1752 had a reduced odds of colon cancer versus those with G alleles [odds ratio (OR) (GA versus GG), 0.63; 95% confidence interval (CI), 0.39–1.01; OR (AA versus GG), 0.33; 95% CI, 0.07–1.65, Ptrend = 0.02]. Similar results were observed for ALOX5 G-1699A [OR (GA versus GG), 0.59, 95% CI, 0.37–0.94; OR (AA versus GG), 0.27, 95% CI, 0.06–1.32, Ptrend = 0.01]. Statistically significant associations with colon cancer were not observed for the other polymorphisms investigated. We have shown for the first time that a haplotype containing ALOX5 G-1752A and G-1699A in a negative regulatory region of the promoter may influence colon cancer risk in Caucasians.

Abbreviations: ALOX, arachidonate lipoxygenase; BMI, body mass index; CI, confidence interval; COX, cyclooxygenase; NSAID, non-steroidal anti-inflammatory drugs; OR, odds ratio


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the human colon, arachidonic acid is metabolized primarily by cycloxygenase (COX, also known as prostaglandin H synthase or PTGS) and arachidonate lipoxygenase (ALOX) enzymes to bioactive lipids (1), which have been implicated in colon cancer risk (24). Metabolism by COX1 and COX2 leads to the synthesis of prostaglandin H2, the precursor of all other prostaglandins (5). Prostaglandins are generated in most cell types and act as autocrine and paracrine mediators of several cell functions including vasomotor tone, vascular permeability, pain generation, febrile response and uterine contractility (5).

In addition, arachidonic acid can be metabolized by ALOX5, ALOX8, ALOX12 and ALOX15 to their corresponding hydroxyeicosatetraenoic acids (HETEs). These can then be further metabolized to leukotrienes (LTs) or lipoxins through additional sequential reactions (6). ALOX products are generated primarily by inflammatory cells and regulate immune inflammatory response, including the activation of neutrophil chemotaxis and trans-endothelial migration and the mediation of allergic inflammation (1,5). While there has been much focus on the carcinogenic effects of the COX enzymes and prostaglandins (7), research is now emerging, which suggests that ALOX and ALOX by-products also play major roles in carcinogenesis in various tissues, including colon (6).

Several lines of evidence suggest that COX and ALOX enzymes are significant in colon carcinogenesis. For example, rapid metabolism of arachidonic acid and elevated levels of COX and/or ALOX enzymes are found in several cancers, including colon cancer (8). In addition, both COX and ALOX enzymes have been shown to stimulate cell proliferation, angiogenesis and metastasis, and to inhibit apoptosis (1). Perhaps the strongest evidence for a role for the COX and ALOX enzymes in carcinogenesis is that several classes of non-steroidal anti-inflammatory drugs (NSAIDs) have been consistently shown to prevent cancer (2,5,9). While there may be several mechanisms by which NSAIDs are anticarcinogenic, the principle mechanisms include the inhibition of COX2 and induction of ALOX15 (2,6).

Several polymorphisms in ALOX and COX genes have been identified, including G-1752A (dbSNP ID: rs6413416), G-1699A (dbSNP ID: rs4986832) and Glu254Lys (dbSNP ID: rs2228065) in ALOX5; Gln261Arg (dbSNP ID: rs1126667) in ALOX12; Leu237Met (dbSNP ID: rs5789) and Val481Ile (dbSNP ID: rs5794) in COX1; and C-645T (dbSNP ID: rs20420) and Val511Ala (dbSNP ID: rs5273) in COX2. Because of the significant role of arachidonic acid metabolism in colon cancer risk, we hypothesized that these polymorphisms could influence susceptibility to colon cancer in Caucasians and African-Americans. To our knowledge, with the exception of COX2 Val511Ala (10), none of these polymorphisms have been studied with respect to colon cancer. We examined these polymorphisms in a case-control study using two distinct control groups, hospital- and population-based, from the greater Baltimore area. We show for the first time that a common haplotype of ALOX5 containing G-1752A and G-1699A, which are both located in a negative regulatory region of the promoter and are in linkage disequilibrium, are associated with a decreased colon cancer risk in Caucasians.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
All cases were recruited between 1992 and 2003 and resided in the greater Baltimore area at the time of recruitment. Of the 436 colon cancer patients at the University of Maryland Hospital and the Baltimore Veterans Affairs hospital in this time period, 293 were found eligible and agreed to participate in this study. Inclusion criteria included being self-reported Caucasian or African-American and being born in the US. Subjects were excluded if, according to self-report, they had other cancers, were infected with HIV, HBV or HCV, were i.v. drug users, were institutionalized or had a mental impairment. All subjects signed informed consent forms and 255 (87%) were administered an epidemiology questionnaire either while in the hospital or subsequently by telephone. Fourteen questionnaires (5%) were answered by subjects' next-of-kin, and interviews were not granted from 23 (8%) of the case subjects. This questionnaire contained no information on NSAID use and limited information about diet and exercise, although body mass index (BMI) was used as a surrogate for diet/exercise in this study. Blood and/or surgical tissue samples were collected at the time of surgery.

Hospital controls were recruited from Family/Internal Medicine, Thoracic Surgery and Pulmonary Clinics between 1998 and 2003. Of the patients screened (n = 1564), 284 were initially found to be eligible. The major causes for exclusion were geography [living outside the study area (35%)], use of immunosuppressants or steroids (10%) or history of other cancers. Of the eligible individuals, 229 (81%) agreed to participate in the study and met the final eligibility criteria. In-person interviews and blood collection were performed at the subjects' convenience.

Population controls (n = 304) were selected from Motor Vehicle Administration records and contacted by mail and then by telephone between 1998 and 2003. Of the 3334 individuals whose telephone numbers were available, 359 were initially found eligible, and 317 of those agreed to participate of which 304 (85% participation rate) met the final eligibility criteria. Major factors contributing to ineligibility for this population included no contact made (63%), history of cancer (9%), death (8%) or language/mental or physical inability (8%). Once again, in-person interviews and blood collection were performed at the subjects' convenience.

Blood was collected when possible from both cases and controls for DNA extraction; however, in some cases when blood was not available, colon tissue was used to obtain DNA for cases. It is very unlikely that a mutation would have occurred at the exact base of one of the SNPs of interest causing an altered genotype; therefore, the potential artifact in archived specimen genotyping is minimal. This protocol was approved by the NCI IRB and the IRBs of all participating institutions.

DNA isolation and genotyping
DNA was isolated from buffy coat or colon tissue, using the Qiagen FlexiGene DNA Kit or the DNeasy Tissue Kit, respectively (Qiagen, Valencia, CA). All genotyping was performed at the National Cancer Institute Core Genotyping Facility, using validated assays that can be found on their website, http://snp500cancer.nci.nih.gov (11). For each SNP, the rate of undetermined calls was between 2 and 5%. Missing genotype data were randomly distributed among subjects, and therefore, will not affect results. Samples that failed to genotype were scored as missing. Ten percent of all samples were genotyped twice for quality control, and concordance was 100%.

Statistical analysis
All analyses, except for that of linkage disequilibrium and haplotype estimation, were performed using Stata 7.0 (Stata, College Station, TX). Age and BMI were compared using the Student's t-test. Because allele frequencies differed by race, all further analyses were stratified by race. Demographic characteristics and allele frequencies were compared using {chi}2 tests. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression adjusting for age (as a continuous variable) and gender. Linkage disequilibrium was estimated using the EH program (12). Haplotype frequencies were estimated separately for Caucasian and African-American cases and controls using PHASE 2.0 (13,14). All analyses were conducted separately for hospital and population controls, and with both control groups combined. In addition, all genotypes in the hospital control group were compared with those in the population control group, using {chi}2 tests and unconditional logistic regression adjusted for age and gender. Because cases were recruited between 1992 and 2003 while controls were recruited between 1998 and 2003, all analyses were performed additionally using the subset of the cases recruited from 1998 to 2003. These analyses yielded similar results to those using the whole case group and, hence, are not presented. Departures from the Hardy–Weinberg equilibrium were evaluated using {chi}2 tests. Each genotype was in Hardy–Weinberg equilibrium in the controls.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Demographic characteristics of the study population are shown in Table I. The gender distribution differed in the cases and controls, as cases had a higher proportion of men than both control groups. Distributions of age, BMI and race were similar in cases and controls. Age and BMI differed by gender. For males, the average age was 66.03 ± 10.48 and average BMI was 27.77 ± 5.38, while for females, the average age and BMI were 62.60 ± 11.09 and 29.07 ± 10.27, respectively. All analyses presented are adjusted for age and gender; however, models presented are not adjusted for BMI, because BMI did not significantly affect the calculated associations.


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Table I. Demographic characteristics of study subjects

 
All analyses were performed with hospital and population controls separately as well as combined. Combined analyses are presented in the manuscript, but analyses stratified by control group can be found in the Supplementary Material Tables I, II and III. {chi}2 tests (Supplementary Material, Table I) and unconditional logistic regression adjusted for age and gender (data not shown) were used to compare genotype frequency in hospital versus population controls. No significant differences were observed between these two control groups when the populations were restricted to either Caucasians or African-Americans. Although not necessarily statistically significant, when our total control population was divided into the hospital- and population-based groups for comparison with cases, we consistently saw similar ORs and trends (Supplementary Material, Tables II and III).

Allele frequencies of the ALOX and COX genes in Caucasian and African-American controls are shown in Table II. Caucasians and African-Americans had different allele frequencies of ALOX5 Glu254Lys and COX2 C-645T and Val511Ala (P < 0.001). For all three SNPs, the variant alleles were nearly absent in Caucasians, but present in ~4–7% of African-American controls, which was also true of cases (data not shown).


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Table II. ALOX and COX allele frequencies in Caucasian and African-American controls

 
Associations with G-1752A, G-1699A and Glu254Lys in ALOX5, Gln261Arg in ALOX12 and colon cancer are shown in Table III (and Supplementary Material, Tables II and III). At position –1752 of ALOX5, Caucasian participants with GA compared with GG had statistically significant reduced odds of colon cancer, which was further reduced in AA individuals (Ptrend = 0.02; Table III). The trend for decreased risk with increasing A alleles was statistically significant, as well. This was also true for position –1699 of ALOX5: Caucasians with either the heterozygous (GA) or the homozygous (AA) genotype also had a reduced risk of colon cancer compared with those with the GG genotype (Ptrend = 0.01). There were no significant associations seen between these polymorphisms and colon cancer risk in African-Americans (Table III); nor between either Glu254Lys in ALOX5 or Gln261Arg in ALOX12 in either Caucasians or African-Americans (P > 0.17 for all). There was significant evidence for interaction between both ALOX5 G-1752A and G-1699A and race with respect to colon cancer risk (Pinteraction < 0.05 for both sites).


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Table III. Association between polymorphisms in ALOX5 and ALOX12 and colon cancer in Caucasians and African-Americans

 
ALOX5 G-1752A and G-1699A polymorphisms were in linkage disequilibrium (P < 0.001). As a first approach to determining whether these polymorphisms together affected colon cancer risk, they were examined in combination. As shown in Table III (and Supplementary Material, Tables II and III), if at least one allele at either –1752 or –1699 was an A, this was associated with a significantly decreased risk of colon cancer in Caucasians. Once again, there was a statistically significant trend for an increasing number of A alleles at either of these sites and colon cancer risk in Caucasians (P = 0.01). There was no significant association found between these SNPs in combination and colon cancer risk in African-Americans, which was expected, given the significant interaction between this SNP combination and race (Pinteraction < 0.05).

Haplotype analyses were conducted for the three ALOX5 SNPs investigated based on an analysis using PHASE 2.0 (Table IV) (13,14). In Caucasians, those with A-A-Glu/G-G-Glu at positions –1752, –1699 and amino acid 254 had a reduced odds ratio for colon cancer compared with those with the most common haplotype, G-G-Glu/G-G-Glu (OR, 0.58; 95% CI, 0.37–0.94). In addition, those who were A-A-Glu/A-A-Glu versus G-G-Glu/G-G-Glu had a non-significant reduced risk of colon cancer (OR, 0.14; 95% CI, 0.02–1.14). Interestingly, there was a significant difference in the overall frequency of common haplotypes between the African-American and Caucasian control groups ({chi}2 = 47.26, P < 0.001). There was no difference between common haplotype frequencies in African-American cases and controls.


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Table IV. Predicted ALOX5 G-1752A, G-1699A, and Glu254Lys diplotype frequencies in African-American and Caucasian cases and controls

 
Associations between colon cancer risk and the Leu237Met and Val481Ile polymorphisms in COX1, and the C-645T and Val511Ala polymorphisms in COX2, were next examined (Supplementary Material, Table IV). The allele frequencies were similar in cases and controls (P > 0.10 for all sites). Because of the small number of individuals with the variant genotypes at each of these sites, homozygous variant genotypes and heterozygotes were combined for all other analyses. There were no significant associations with colon cancer risk observed for these polymorphisms, although there was a small non-significant increased risk of colon cancer in subjects with a 237Met allele of COX-1: OR (Leu/Met or Met/Met versus Leu/Leu, 2.10; 95% CI 0.98–4.48).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Metabolism of arachidonic acid by ALOX and COX enzymes has been shown to contribute to the carcinogenic processes in multiple tissues, including the colon (1). We investigated whether polymorphisms in the genes that encode these enzymes also contributed to colon cancer risk. We have shown for the first time that the G-1752A and G-1699A polymorphisms in ALOX5 are significantly associated with decreased colon cancer risk in Caucasians, but not African-Americans, while Glu254Lys in ALOX5, Gln261Arg in ALOX12, Leu237Met and Val481Ile in COX1 and C-645T, and Val511Ala in COX2 do not appear to be associated with colon cancer risk.

ALOX5 catalyzes the first committed steps for the synthesis of all LT (15). It was shown to be over-expressed in cancer cells and human tumors, and to promote cancer cell growth and angiogenesis (1). While we found no association between the Glu254Lys polymorphism and colon cancer risk, we have shown that G to A polymorphisms at –1752 and –1699 are in linkage disequilibrium and are significantly associated with a decreased risk of colon cancer in Caucasians when analyzed separately, in combination or as part of common ALOX5 haplotypes. We were not surprised to find that ALOX5 G-1752A and G-1699A are in linkage disequilibrium, because they are only 53 bp apart. We cannot determine whether one or both of the polymorphisms are functional based on these data, but further studies are warranted to investigate these findings. Because both positions are in a negative regulatory region of the promoter (16), we hypothesize that these polymorphisms could enhance binding to this site, thus decreasing transcription of ALOX5, which, in turn, could lower cancer risk by quantitatively decreasing enzymatic activity.

Previously, a polymorphism had been identified in the core promoter of the ALOX5 gene, which is characterized by the deletion or addition of consensus Sp1 (-GGGCGG) and Egr-1 (-GCGGGGGCG-) binding motifs 176–147 bp upstream from the ATG translation start site (17,18). Each of the variant alleles binds the Sp1 and Egr-1 proteins, and the transactivation of both Sp1 and Egr-1 is proportional to the number of Sp1/Egr-1 consensus binding sites within the GC-rich sequence (17,18). Several groups examined associations between these polymorphisms and various diseases associated with inflammation, including Alzheimer's disease (19), asthma (20,21) and atherosclerosis (22). While some studies found null or modest results, Dwyer et al. (22) found that having a common variant genotype that alters binding to Sp1 led to an increased risk of atherosclerosis, suggesting decreased ALOX5 expression could lead to higher disease risk.

While associations between –1752 and –1699 ALOX5 polymorphisms and reduced colon cancer risk were observed in our Caucasian sample population, this was not seen in our African-American sample population. Stratifying the study population by race could have led to spurious results due to small sample sizes. On the other hand, it is biologically plausible that Caucasians and African-Americans have different colon cancer etiologies. Of all races studied, African-Americans have the highest incidence of and mortality from colon cancer (23). It is possible that some individual or combination of genetic and/or environmental factors exist in African-Americans that counteract the potential decrease in colon cancer risk attributable to these ALOX5 polymorphisms, resulting in their not having an observable effect on colon cancer risk.

ALOX12 generates 12-S-HETE, which has been shown to significantly contribute to carcinogenesis by promoting invasion, metastasis and angiogenesis (24). The Gln261Arg polymorphism in ALOX12 is in the lypoxygenase domain, a highly conserved region of the protein (25), but the functional signifiance of this residue is unclear. We found no association between this polymorphism in ALOX12 and colon cancer risk. In a previous study, the Gln261Arg polymorphism was associated with bipolar disorder in Brazilian patients, suggesting that the polymorphism may be functional itself or linked to a functional polymorphism (25).

An abundance of evidence supports a role for COX2 in colon cancer risk. COX2 is often over-expressed in human colon cancer and the inhibition of COX2 has been shown to reduce colon cancer development in both humans and animals (7). We examined the possible role of two polymorphisms in COX2 and colon cancer risk, and found no associations for either C-645T or Val511Ala. To our knowledge, the functionality of the C-645T polymorphism in the COX2 promoter region has not yet been examined; however, two groups have analyzed the functionality of the Val511Ala polymorphism. Fritsche et al. (26) expressed the variant Val511Ala proteins and compared the metabolism of COX2 substrates, including arachidonic acid, but found no differences in activity. Lin et al. (10) also found no difference in kinetic parameters of the two variants. However, Lin et al. found that residue 511 lies inside a tightly packed hydrophobic pocket adjacent to the active site. They found the allele frequency of this polymorphism was 4% in African-Americans and 0% in Caucasians, which is consistent with our study. Lin et al. also found that the Ala511 allele was protective against colon cancer in three separate African-American populations; however, the results were not statistically significant, again, consistent with our study.

Traditionally, COX1 has not been thought to play a significant role in colon cancer risk; however, recent data suggest that it may be involved. Several studies suggest that COX1 activity is critical for the development of colon cancer, perhaps through COX2 induction (9). In addition, aspirin use at doses too low to sustain COX2 inhibition, but which can inhibit COX1, are associated with a decreased colon cancer risk in epidemiology studies (27,28). Similar to our findings, Halushka et al. (29) found the variant alleles of both Leu237Met and Val481Ile were present in one out of 76 chromosomes. Ulrich et al. (30) also searched for COX1 variants and found the Leu237Met polymorphism, but not Val481Ile. The allele frequency of Leu237Met was 4% in two Caucasian populations and 0% in an African-American population. Although we saw no significant association between either polymorphism and colon cancer risk, we cannot rule out potential associations that are low in magnitude. With the low allele frequency of the COX1 and COX2 polymorphisms seen in our study and that of others, a large prospective study may be more successful at determining risk, particularly if the increased risk is low enough that smaller studies may not have a high enough power to detect it.

One of the major strengths of this study is the use of two control groups. It has been suggested that the use of several control groups increases the validity of inferences drawn, because it gives greater confidence that the apparent association between exposure and disease is real (31). Furthermore, population and hospital controls each have unique advantages and disadvantages. Advantages of population controls include that controls are drawn from the same catchment population as the cases and the distribution of exposures in controls can be extrapolated back to the population (32). In contrast, hospital controls might not represent the catchment population as well, because only a subset of the population may go to specific hospitals from where the cases are recruited (32). On the other hand, hospital controls can often provide the same quality of information as cases versus population controls, who may be less motivated to participate and may have different recall biases than cases do (32). The use of both hospital and population control groups adds strength to our conclusions. In our study, SNP distributions were similar in both control groups, which suggests that potential weaknesses of each control group were minimal. Furthermore, although not always statistically significant, when our total control population was divided into the hospital- and population-based control groups for analyses with cases, we consistently saw similar ORs and trends, again, strengthening our findings.

A limitation of our study is that we are missing information about certain risk factors, such as diet, exercise and NSAID use, which have been consistently associated with a decreased risk of colon cancer (9). There is currently no evidence that NSAIDs affect ALOX5 or ALOX12, therefore, this should have no impact on the association between ALOX5 and ALOX12 polymorphisms and colon cancer risk. Furthermore, because NSAID use is not likely related to the ALOX or COX genotypes, it should not be a confounder of any of the polymorphism/colon cancer risk associations in this study. However, in the future, it may be interesting to determine more conclusively if NSAID use affects the associations seen between these polymorphisms and colon cancer in other populations.

In summary, haplotypes containing the G-1752A and G-1699A polymorphisms in ALOX5 are significantly negatively associated with colon cancer risk in Caucasians; and individual SNP analyses were consistent with the haplotype analysis. There were no associations seen for Glu254Lys in ALOX5; Gln261Arg in ALOX12; Leu237Met and Val481Ile in COX, and C-645T and Val511Ala in COX2. Future epidemiological studies should be performed to confirm these results and mechanism-based laboratory studies should be conducted to further elucidate the specific functions of these polymorphisms.


    Acknowledgments
 
The authors thank Jackie Lavigne, Leah Mechanic and Stefan Ambs for their intellectual and editorial input and Dorothea Dudek for her editorial assistance. We would also like to thank Raymond Jones, John Cottrell, Donna Perlmutter, Audrey Salabes, Bonnie Cooper, Christopher Loffredo, Peter Shields, Leoni Leondaridis, Glennwood Trivers and Brenda Boersma for their contributions to this study.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 2, 2004; revised July 23, 2004; accepted August 2, 2004.