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. Author manuscript; available in PMC: 2014 May 1.
Published in final edited form as: Genes Chromosomes Cancer. 2013 Feb 12;52(5):437–449. doi: 10.1002/gcc.22042

Genetic Variation in the Lipoxygenase Pathway and Risk of Colorectal Neoplasia

Sarah E Kleinstein 1,2, Laura Heath 1, Karen W Makar 1, Elizabeth M Poole 1,3, Brenna L Seufert 1, Martha L Slattery 4, Liren Xiao 1, David J Duggan 5, Li Hsu 1, Karen Curtin 4, Lisel Koepl 1, Jill Muehling 5, Darin Taverna 5, Bette J Caan 6, Christopher S Carlson 1, John D Potter 1,2,7, Cornelia M Ulrich 1,2,8,*
PMCID: PMC3698944  NIHMSID: NIHMS461690  PMID: 23404351

Abstract

Arachidonate lipoxygenase (ALOX) enzymes metabolize arachidonic acid to generate potent inflammatory mediators and play an important role in inflammation-associated diseases. We investigated associations between colorectal cancer risk and polymorphisms in ALOX5, FLAP, ALOX12, and ALOX15, and their interactions with non-steroidal anti-inflammatory drug (NSAID) use. We genotyped fifty tagSNPs, one candidate SNP, and two functional promoter variable nucleotide tandem repeat (VNTR) polymorphisms in three US population-based case-control studies of colon cancer (1424 cases/1780 controls), rectal cancer (583 cases/775 controls), and colorectal adenomas (485 cases/578 controls). Individuals with variant genotypes of the ALOX5 VNTR had decreased risk of rectal cancer, with the strongest association seen for individuals with one or more alleles of >5 repeats (wildtype=5, OR>5/≥5=0.42, 95% CI 0.20-0.92; p=0.01). Four SNPs in FLAP (rs17239025), ALOX 12 (rs2073438), and ALOX15 (rs4796535 and rs2619112) were associated with rectal cancer risk at p≤0.05. One SNP in FLAP (rs12429692) was associated with adenoma risk. A false discovery rate (FDR) was applied to account for false positives due to multiple testing; the ALOX15 associations were noteworthy at 25% FDR. Colorectal neoplasia risk appeared to be modified by NSAID use in individuals with variant alleles in FLAP and ALOX15. One noteworthy interaction (25% FDR) was observed for rectal cancer. Genetic variability in arachidonate lipoxygenases may affect risk of colorectal neoplasia, particularly for rectal cancer. Additionally, genetic variability in FLAP and ALOX15 may modify the protective effect of NSAID use against colorectal neoplasia.

INTRODUCTION

Inflammation plays a key role in colorectal carcinogenesis. Two essential and competing pathways that are involved in modulating the inflammatory response are the leukotriene and prostaglandin pathways, both of which use the same primary precursor, arachidonic acid (AA) (Romano and Claria, 2003). Associations between genetic polymorphisms in prostaglandin synthesis enzymes and colorectal neoplasia risk have been established in multiple studies (Lin et al., 2002; Ulrich et al., 2002, 2004, 2005; Goodman et al., 2004; Koh et al., 2004; Poole et al., 2007, 2010; Cross et al., 2008; Liu et al., 2011), and genetic polymorphisms in a key leukotriene pathway gene family, the arachidonic lipoxygenases (ALOXs), have more recently been implicated in various cancers and inflammatory diseases such as asthma, bone loss, and atherosclerosis (Poole et al., 2005; Ichikawa et al., 2006; Mullin et al., 2007; Paganelli et al., 2007; Tranah et al., 2008; Bhattacharya et al., 2009; Krönke et al., 2009; Feng et al., 2010; Lindley et al., 2010; Liu et al., 2010; Gertow et al., 2011). A primary function of the ALOXs is to convert AA into leukotrienes, a class of paracrine hormones involved in the inflammatory response, as well as other inflammation-mediating eicosanoids which have been implicated in a variety of inflammatory diseases, including colorectal cancer (Funk, 2001; Nie and Honn, 2002).

The bioactive compounds generated by genes in the lipoxygenase pathway result in a variety of biologic activities important for carcinogenesis. Both arachidonate 5-lipoxygenase (ALOX5) and 12-lipoxygenase (ALOX12) have been described as pro-carcinogenic. In particular, ALOX5 and the 5-lipoxygenase-activating protein (FLAP, also known as ALOX5AP), are up-regulated in colon, esophageal, breast, prostate, and pancreatic cancers, and downstream metabolites of the ALOX5 cascade enhance cell proliferation and survival (Anderson et al., 1998; Avis et al., 2001; Shureiqi and Lippman, 2001; Hennig et al., 2002; Tong et al., 2002; Nielsen et al., 2003; Hoque et al., 2005; Jiang et al., 2006). ALOX12 converts AA to 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which increases expression of pro-inflammatory cytokine genes such as tumor necrosis factor-α (Chakrabarti et al., 2009). ALOX15, an oxidizing enzyme that converts AA to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), has anti-carcinogenic properties; it both increases apoptosis and decreases cancer cell proliferation (Feng et al., 2010). The under-expression of ALOX15 has been associated with the development of cancer and osteoporosis, while over-expression has been linked to asthma and atherosclerosis (Tranah et al., 2008; Krönke et al., 2009; Feng et al., 2010; Lindley et al., 2010; Liu et al., 2010; Gertow et al., 2011).

Previous studies point to functional implications of genetic variation in the lipoxygenase pathway. A rare ALOX15 coding single nucleotide polymorphism (SNP, T560M) that has been implicated in coronary artery disease resulted in reduced enzyme activity in vitro (Schurmann et al., 2011), whereas promoter-region polymorphisms in both ALOX15 (−292C>T) and ALOX5 (a variable nucleotide tandem repeat (VNTR) (−176(GGGCGG)2-8)) were associated with transcription modification in vitro (In et al., 1997; Silverman and Drazen, 2000; Wittwer et al., 2006). In an earlier study, we noted evidence for interactions between this ALOX5 VNTR and a previously identified functional COX-2 promoter polymorphism (−765G>C) that modulated risk of adenomatous polyps (Poole et al., 2006). In addition, a candidate SNP in the negative regulatory region of the ALOX5 promoter, rs4986832 (−1700 G>A), has previously been associated with a decreased risk of colon cancer (Goodman et al., 2004).

Long-term NSAID use reduces the risk of colorectal neoplasia and other diseases (Taketo, 1998; Giovannucci, 1999; Baron et al., 2003; Sandler et al., 2003; Ulrich et al., 2006; Chan et al., 2012) by targeting COX-1 and COX-2 to reduce the prostaglandin-induced inflammation response (Vane, 1971; Ulrich et al., 2006). However, prescribing aspirin and other NSAIDs as chemopreventive agents needs to be balanced with risk of adverse effects, such as bleeding (Ulrich et al., 2006). As the leukotriene and prostaglandin pathways are in competition, ALOX gene polymorphisms might indirectly interact with NSAID use to alter the protective effect on colon cancer, which would have pharmacogenetic implications for prescribing NSAIDs long-term for some individuals. Furthermore, in vitro studies show that NSAIDs induce apoptosis in colon cancer cells via up-regulation of ALOX15 (Shureiqi et al., 2000; Shureiqi and Lippman, 2001), suggesting polymorphisms in ALOX15 may directly interact with NSAID use.

We hypothesized that genetic variability in ALOX5, FLAP, ALOX12, and ALOX15 influences colorectal carcinogenesis by altering the balance between prostaglandin and leukotriene synthesis and/or by modulating protein expression in the lipoxygenase pathway, with potential implications for effect modification by NSAID use. We present a comprehensive investigation of tagSNPs and functional candidate polymorphisms in the lipoxygenase enzyme pathway across a continuum of colorectal carcinogenesis by including adenoma, rectal cancer, and colon cancer populations. We also identify potential interactions between NSAID use and lipoxygenase polymorphisms that modify colorectal cancer risk.

MATERIALS AND METHODS

Study Populations

Adenoma study

Colorectal adenoma cases (n=485) and polyp-free controls (n=578) were recruited through a large multi-clinic gastroenterological practice in the Twin Cities area of Minnesota (Potter et al., 1996). In brief, patients were identified with a first diagnosis of adenomatous or hyperplastic polyp at the time of colonoscopy; only adenomatous cases were included for further study due to small sample sizes of hyperplastic cases. Eligible participants were aged 30-74 years, with a first colorectal adenoma diagnosis from 1991-1994, no known genetic syndrome associated with increased colon neoplasia risk, and no individual history of cancer (except non-melanoma skin cancer), prior colorectal polyps, or inflammatory bowel disease. All participants underwent colonoscopy. The participation proportion among patients receiving colonoscopy was 68%.

Colon and rectal cancer studies

Colon cancer cases (n=1424) and controls (n=1780) and rectal cancer cases (n=583) and controls (n=775) were recruited from the northern California Kaiser Permanente Medical Care Program (KPMCP), Utah, and the Twin Cities metropolitan area of Minnesota (colon cancer only) (Slattery et al., 1997; Slattery et al., 2003a). Eligible cases were aged 30-79, with no previous diagnosis of colorectal cancer, familial adenomatous polyposis, ulcerative colitis, or Crohn’s disease. Colon cancer cases were first diagnosed from 1991-1994; rectal cancer cases were first diagnosed from 1997-2001. Participation proportions among contacted colon cancer cases were 76% and 64% for controls. Participation proportions among contacted rectal cancer cases were 73% and, for controls, 69%.

Questionnaire Data and Blood Collection

Information on use of NSAIDs, lifestyle factors and diet, anthropometry, demographics, and medical information (including family history of cancer) was obtained by questionnaire, as described previously (Potter et al., 1996; Slattery et al., 1997, 2003a; Ulrich et al., 1999). Buffy coats were obtained within 24 hours of venous blood collection for genomic DNA extraction, quantitation, and genotyping analyses.

SNP Selection and Genotyping

An identical tagSNP selection and genotyping procedure was used in all three studies. The coding regions and 2 kb beyond the 5′ and 3′ ends of FLAP, ALOX12 and ALOX15 have been resequenced in 23 individuals of European descent by Seattle SNPs (http://pga.gs.washington.edu). TagSNPs were identified in the resequenced individuals using the Linkage Disequilibrium (LD) Select algorithm (Carlson et al., 2004), with MAF=4% and r2=0.9. Calculations were performed in QUANTO (Gauderman and Morrison, 2006) to determine that for SNPs with MAF>4% and reasonable effect sizes, sample sizes were sufficient and adequately powered in the colon and rectal cancer populations, and more weakly powered in the adenoma population. Thirty-two tagSNPs in FLAP, 17 in ALOX12, and 21 in ALOX15 were successfully converted to the Illumina™ GoldenGate genotyping platform as described below. There were no resequencing data available for ALOX5, thus only one candidate SNP (rs4986832, −1700G>A) was chosen for inclusion and was genotyped by Taqman assay (Applied Biosystems) at the Fred Hutchinson Cancer Research Center (FHCRC).

Two variable nucleotide tandem repeats (VNTRs) were also sequenced: the ALOX5 VNTR promoter polymorphism [−176(GGGCGG)2-8] and the FLAP poly(A) promoter repeat [−169poly(A)]. The VNTRs were genotyped at FHCRC using GeneScan on an ABI 3130xl, based on previously-published protocols (Sayers et al., 2003; Poole et al., 2006). Briefly, multiplex PCR reactions consisted of 40ng genomic template DNA, 1.5mmol/L MgCl2, 1x PCR Gold Buffer (Applied Biosystems), 0.5 units of Amplitaq Gold Polymerase (Applied Biosystems), 200nmol/L each of oligonucleotide primers ALOX5sp1-F 5′-6FAM-AGGAACAGACACCTCGCTGAGGAGAG-3′, ALOX5sp1-R 5′GAGCAGCGAGCGCCGGGAGCCTCGGC-3′, ALOX5AP-F: 5′ VICCGTGCTCCTCTGCCAAGCCCTGCTTC-3′, and ALOX5AP-R02: 5′-GCTCTGCCTCCAGCTGCACAACCTG-3′, 150μmol/L each of dATP, dCTP and dTTP, 75μmol/L each of dGTP and 7-deaza-2V-dGTP (Roche Diagnostics GmbH, Mannheim, Germany), and 8% (v/v) DMSO (Sigma, St. Louis, MO) in 5 uL. Cycling was as follows: 96°C for 7 minutes, 30 cycles of: 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, 72°C for 7 minutes. The observed 6FAM-labeled ALOX5 amplicon lengths ranged from 254bp (2 6-bp repeats) to 290bp (8 6-bp repeats) and VIC-labeled FLAP amplicon was either 212bp (19A) or 216bp (23A).

Quality Control

All tagSNPs were genotyped using Illumina™ GoldenGate bead-based genotyping technology at the Translational Genomics Research Institute (TGen, Phoenix, Arizona). Intraplate and interplate replicates (5%) were included for all plates and batches, as well as blinded duplicates. Genotype data from 30 CEPH trios (Coriell Cell Repository, Camden, NJ) that had been genotyped by the HapMap project were used to confirm reliability and reproducibility of the genotyping assays. Quality-control (QC) checks based on Illumina metrics were performed as described previously (Levine et al., 2010). Genotypes were excluded from analyses by TGen if any of the following were true: GenTrain Score <0.4, 10% GC Score <0.25, AB T Dev >0.1239, Call Frequency <0.85, Replicate Errors >2, P-P-C Errors >2. Further exclusions were made for SNPs that had <85% concordance with blinded or non-blinded duplicates and for Hardy-Weinberg Equilibrium (HWE) p-values <0.0001. A total of twenty SNPs failed QC under the guidelines listed previously, thirteen of which were in ALOX15. Subsequent mapping of an ALOX15 pseudogene on chromosome 17 accounted for the ALOX15 HWE p<0.001 failed SNPs. 28 FLAP SNPs, 14 ALOX12 SNPs, 8 ALOX15 SNPs, the ALOX5 SNP, and both VNTRs passed QC measures and were included in the analyses (Supplementary Table 1). When two SNPs were observed to be in high LD (r2≥0.90) in our study population, only one SNP was presented, given that the two SNPs would likely capture the same information.

Statistical Data Analysis

All analyses were conducted using SAS 9.1/9.2 software (SAS Institute, Cary, NC). Logistic regression analysis was used to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs). A co-dominant model was used as long as each cell contained ≥5 individuals; otherwise, a dominant model was analyzed. In a primary analysis examining associations between polymorphisms and colorectal neoplasia risk, trends were also analyzed via a log-additive model. The main effects analyses were adjusted for age, sex, and study site (if applicable) only, as other covariates (such as BMI, calcium intake, smoking, dietary fiber, study site, and activity) did not have significant impacts on the risk ratios. We would not expect, a priori, to see associations between health-behavior-related covariates and the candidate genes under study. The analyses were restricted to individuals who self-reported as non-Hispanic white (representing 97%, 91% and 87% in the adenoma, colon and rectal cancer studies, respectively) to limit population stratification.

In the secondary analysis exploring the interaction between the genotyped polymorphisms with NSAID use on colorectal neoplasia risk, multivariate adjustments were used, controlling for age, sex, body mass index (BMI), dietary intake of fiber (gm/day), alcohol (gm/day), energy (calories/day), hours/wk vigorous physical activity, and smoking (cigarettes/day). NSAID use was analyzed comparing regular use to non-regular use (≥1/wk v. ≤1/wk in the adenoma study, ≥3/wk v. ≤3/wk in colon and rectal studies). Regular NSAID use was inversely associated with colon cancer, rectal cancer, and adenoma risk (Bigler et al., 2001; Slattery et al., 2004b) (ORcolon=0.64, 95% CI 0.55-0.74, p<0.0001; ORrectal=0.67, 95% CI 0.54-0.85, p=0.0006; ORadenoma=0.65, 95% CI 0.49-0.85, p=0.0018; adjusted as stated above). Possible effect modification was evaluated using the likelihood ratio test comparing models with and without the multiplicative genotype x NSAID interaction term(s) (with two degrees of freedom (d.f.) for codominant model, 1 d.f. for dominant model).

The ALOX5 VNTR wildtype genotype (the referent) was defined as five repeats of the six bp motif (-GGGCGG-) in both alleles (noted as 5/5 in Table 2). Individuals with <5 repeats in one or both alleles (<5/≤5) were grouped together, as were individuals with >5 repeats in one or both alleles (>5/≥5). The FLAP VNTR is a poly (A) microsatellite containing either 19 (wildtype) or 23 (variant) A’s.

Table 2.

Associations between polymorphisms in lipoxygenase pathway and risk of colorectal neoplasia

Colon cancer
Rectal cancer
Adenomas
SNPs Genotype Cases/
Controls
OR 95% CI pa ptrend Cases/
Controls
OR 95% CI pa ptrend Cases/
Controls
OR 95% CI pa ptrend
ALOX5

VNTR 5/5b (ref) 979/1188 1.00 430/518 1.00 331/372 1.00
<5/≤5 415/530 0.96 (0.82-1.12) 171/274 0.76 (0.60-0.95) 154/200 0.86 (0.65-1.13)
>5/≥5 26/50 0.63 (0.39-1.01) 0.14 NA 9/26 0.42 (0.20-0.92) 0.01 NA 18/19 0.92 (0.45-1.87) 0.54 NA
rs4986832 GG 1011/1242 1.00 421/521 1.00 336/389 1.00
−1700 G>A AG 372/489 0.94 (0.80-1.10) 149/226 0.82 (0.64-1.05) 135/178 0.87 (0.66-1.16)
AA 40/45 1.09 (0.70-1.68) 0.64 0.66 13/27 0.60 (0.31-1.18) 0.11 0.04 12/15 0.94 (0.42-2.14) 0.63 0.40

FLAP

rs12429692 AA 795/1012 1.00 313/438 1.00 263/318 1.00
2439 A>T AT 529/651 1.02 (0.88-1.19) 231/291 1.11 (0.89-1.40) 177/236 0.91 (0.70-1.19)
TT 97/113 1.11 (0.84-1.48) 0.75 0.50 39/43 1.26 (0.80-2.00) 0.46 0.21 42/27 2.05 (1.20-3.53) 0.01 0.20
rs17239025 GG 1266/1607 1.00 513/707 1.00 431/528 1.00
30185 G>C GC/CCc 152/163 1.18 (0.93-1.49) 0.17 NA 70/68 1.43 (1.01-2.04) 0.05 NA 51/54 1.23 (0.80-1.89) 0.34 NA

ALOX12

rs2073438 GG 704/912 1.00 301/416 1.00 238/303 1.00
639 G>A AG 608/712 1.11 (0.96-1.28) 251/292 1.20 (0.96-1.50) 197/231 1.05 (0.80-1.38)
AA 104/149 0.91 (0.69-1.19) 0.21 0.74 30/64 0.66 (0.42-1.04) 0.02 0.85 47/47 1.23 (0.77-1.95) 0.68 0.42

ALOX15

rs4796535 GG 1252/1540 1.00 498/692 1.00 433/546 1.00
1351 G>A AG/AAc 161/226 0.87 (0.70-1.08) 0.22 NA 85/83 1.43 (1.03-1.97) 0.03 d NA 50/63 0.96 (0.63-1.45) 0.83 NA
rs2619112 GG 409/513 1.00 143/230 1.00 136/164 1.00
9562 G>A AG 721/868 1.03 (0.88-1.22) 316/371 1.37 (1.06-1.77) 244/295 0.98 (0.73-1.32)
AA 286/392 0.92 (0.75-1.12) 0.43 0.46 120/172 1.13 (0.83-1.55) 0.05 d 0.34 102/123 1.06 (0.74-1.53) 0.89 0.77
a

global p-value from co-dominant model; ptrend from log-additive model

b

Genotype refers to the number of repeats per allele; wildtype = 5 repeats in both alleles (the referent)

c

the global p-value for the dominant rather than co-dominant model is provided when any cell < 5 (p-trend not applicable)

d

Italics underlined = noteworthy at 25% FDR

All statistical tests were two-sided. The initial statistical significance level was set at a cutoff of α=0.05. Since our study is investigating variation in candidate genes that were hypothesized a priori to affect colorectal carcinogenesis, conventional multiple comparisons tests were not applied due to the risk of type II errors (the rejection of true associations) (Rothman, 1990; Streiner and Norman, 2011). Instead, along with presenting uncorrected p-values, the less conservative false discovery rate (FDR) analyses were conducted using the Benjamini and Hochberg (B&H) method (Benjamini and Hochberg, 1995; Benjamini et al., 2001). In brief, rather than protecting against type I errors, the B&H method allows for false positives in the process of discovering true positives at or below a pre-specified FDR, which is the expected proportion of false positives among all the hypotheses determined to be “noteworthy” associations, as described in detail previously (Coghill et al., 2011). For example, 75% of SNPs that are identified as noteworthy by FDR at the 25% level are likely true positives. We set the B&H cutoff for noteworthy SNPs at the 25% level for both main effect and NSAID interaction analyses. FDR analyses were conducted on a gene by gene basis for the tagSNPs and were not conducted for the candidate polymorphism or VNTRs in ALOX5 and FLAP. Tables 2 and 3 include all associations having initial uncorrected p-values≤0.05, and “noteworthy” SNPs below the 25% FDR. Complete results are in Supplementary Tables 2 and 3.

Table 3.

Interactions between lipoxygenase tagSNPs, NSAID use and risk of colorectal neoplasia

Colon Cancer NSAID Use
Rectal Cancer NSAID Use
Adenoma NSAID Use
Non-Regular Regular Non-Regular Regular Non-Regular Regular



SNP name Genotype OR 95% CI OR 95% CI pint OR 95% CI OR 95% CI pint OR 95% CI OR 95% CI pint
FLAP

rs17239025 GG 1.00 . 0.60 (0.51-0.71) 1.00 . 0.67 (0.53-0.85) 1.00 . 0.61 (0.50-0.80)
30185 G>C GC/CCa 0.96 (0.71-1.29) 1.03 (0.70-1.52) 0.02 1.38 (0.88-2.16) 1.06 (0.58-1.93) 0.73 1.23 (0.6-2.4) 0.84 (0.50-1.60) 0.82
rs9508832 GG 1.00 0.58 (0.44-0.76) 1.00 0.64 (0.43-0.95) 1.00 0.55 (0.34-0.89)
4527 G>A AG 0.95 (0.77-1.17) 0.62 (0.49-0.79) 0.88 (0.64-1.22) 0.76 (0.54-1.08) 0.68 (0.45-1.02) 0.46 (0.30-0.73)
AA 0.92 (0.71-1.18) 0.61 (0.44-0.84) 0.72 1.10 (0.72-1.67) 0.38 (0.23-0.63) 0.02 0.82 (0.49-1.35) 0.45 (0.24-0.84) 0.76
rs9315053 TT 1.00 0.64 (0.51-0.80) 1.00 0.77 (0.55-1.07) 1.00 0.61 (0.41-0.91)
29380 T>G GT 1.03 (0.85-1.24) 0.67 (0.53-0.84) 1.00 (0.74-1.36) 0.73 (0.52-1.02) 0.73 (0.50-1.05) 0.45 (0.30-0.69)
GG 0.95 (0.71-1.28) 0.49 (0.32-0.76) 0.68 1.58 (0.97-2.59) 0.44 (0.23-0.81) 0.03 0.75 (0.42-1.34) 0.45 (0.20-1.02) 1.00
rs4075692 GG 1.00 0.64 (0.48-0.85) 1.00 0.61 (0.39-0.94) 1.00 0.63 (0.37-1.05)
13604 G>A AG 1.00 (0.80-1.24) 0.64 (0.50-0.82) 0.85 (0.60-1.20) 0.75 (0.52-1.08) 0.66 (0.43-1.00) 0.46 (0.29-0.72)
AA 0.99 (0.77-1.29) 0.61 (0.44-0.83) 0.97 0.97 (0.64-1.47) 0.40 (0.25-0.64) 0.03 0.82 (0.50-1.33) 0.37 (0.20-0.67) 0.50
rs9551960 AA 1.00 0.57 (0.44-0.74) 1.00 0.53 (0.36-0.79) 1.00 0.75 (0.45-1.23)
7202 G>A AG 0.99 (0.81-1.21) 0.71 (0.56-0.89) 0.81 (0.58-1.12) 0.51 (0.36-0.73) 1.20 (0.81-1.78) 0.65 (0.42-1.01)
GG 0.95 (0.73-1.24) 0.55 (0.40-0.76) 0.35 0.69 (0.46-1.05) 0.82 (0.51-1.32) 0.05 1.21 (0.72-2.03) 0.73 (0.42-1.29) 0.61

ALOX15

rs2619112 CC 1.00 0.61 (0.46-0.81) 1.00 0.58 (0.37-0.90) 1.00 0.70 (0.42-1.17)
9562 C>T CT 1.01 (0.82-1.25) 0.68 (0.54-0.86) 1.10 (0.79-1.55) 1.00 (0.69-1.43) 0.97 (0.64-1.46) 0.65 (0.42-1.02)
TT 0.93 (0.71-1.20) 0.53 (0.39-0.73) 0.71 1.30 (0.86-1.97) 0.49 (0.31-0.80) 0.01 b 1.30 (0.78-2.17) 0.55 (0.31-0.98) 0.38
rs2664593 CC 1.00 0.68 (0.56-0.82) 1.00 0.75 (0.57-1.00) 1.00 0.55 (0.38-0.78)
−189 G>C CG 1.14 (0.94-1.39) 0.64 (0.50-0.81) 1.07 (0.79-1.45) 0.61 (0.43-0.86) 0.95 (0.66-1.37) 0.61 (0.39-0.96)
GG 1.31 (0.86-1.99) 0.95 (0.58-1.56) 0.48 0.93 (0.45-1.92) 0.39 (0.14-1.14) 0.38 0.19 (0.06-0.57) 0.93 (0.26-3.28) 0.04
a

the p-interaction for the dominant model is provided when any cell < 5

b

Italics underlined = noteworthy at 25% FDR

RESULTS

Characteristics of the Study Populations

Characteristics of the study populations are shown in Table 1. Briefly, the adenoma study participants were youngest on average, while the colon cancer study participants were oldest on average. Cases of all three study populations were more likely to be male, and controls in all three populations were more likely to be regular users of NSAIDs.

Table 1.

Characteristics of the three study populations

Colon cancer study Rectal cancer study Adenoma study

Cases
(N=1424)
Controls
(N=1780)
p-
value
Cases
(N=583)
Controls
(N=775)
p-
value
Cases
(N=485)
Controls
(N=578)
p-value
Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD)
Age (years) 65.2 (9.7) 65.1 (10.3) NAa 62.3 (10.8) 62.6 (10.5) NAa 58 (9.6) 52.9 (11.0) <0.01
Location a N (%) N (%) N (%) N (%) N (%) N (%)
Proximal 688 (49.6) NA NA NA NA NA 104 (21.6) NA NA
Distal 700 (50.4) NA NA NA 300 (62.4) NA
Rectal NA NA 583 775 77 (16.0) NA
Sex
Male 797 (56.0) 946 (53.2) NAa 346 (59.4) 428 (55.2) NAa 304 (62.7) 227 (39.3) <0.01
Female 627 (44.0) 834 (46.9) 237 (40.7) 347 (44.8) 181 (37.3) 351 (60.7)
Study Site
Kaiser Northern California 617 (43.3) 647 (36.4) <0.01 349 (59.9) 449 (57.9) 0.48 NA NA NA
Minnesota 565 (39.7) 791 (44.4) NA NA 485 (100) 578 (100)
Utah 242 (17.0) 342 (19.2) 234 (40.1) 326 (40.1) NA NA
Regular Use of NSAIDs b
Yes 468 (33.2) 748 (42.2) <0.01 221 (38.1) 359 (46.6) <0.01 180 (37.1) 257 (44.5) 0.02
No 943 (66.8) 1023 (57.8) 359 (61.9) 412 (53.4) 305 (62.9) 321 (55.6)
a

Matching factors

b

Regular NSAID use defined as at least 1×/week for adenoma stud,y and at least 3×/week for ≥1 month within 2 years of referent date for colon and rectal cancer studies

Genetic Variation in ALOX5

The ALOX5 functional VNTR polymorphism was associated with a significantly lower risk of rectal cancer for individuals carrying an allele with any number of repeats other than wildtype (5 repeats) (Table 2) (OR<5/≤5 v. 5/5=0.76; 95% CI 0.60-0.95; OR>5/≥5 v. 5/5=0.42; 95% CI 0.20-0.92; global p=0.01). The same (though statistically non-significant) pattern of lower risk in individuals with at least one non-wildtype allele was seen in both colon cancer and adenoma.

For the ALOX5 candidate SNP rs4986832 (−1700G>A), individuals who carried a variant allele had a borderline reduced risk of rectal cancer relative to individuals with the wildtype genotype (ORhet v. wt=0.82; 95% CI 0.64-1.15, ORhzv v. wt=0.60; 95% CI 0.31-1.18; global p=0.11). However, carriage of the variant allele showed a statistically significant dose-response (p-trend=0.04). Because these analyses focused on pre-hypothesized candidate/functional variants, no FDR calculations were applied. There were no statistically significant associations between the candidate polymorphism and colon cancer or adenoma risk, and there were no statistically significant NSAID interactions.

Genetic Variation in FLAP

Two SNPs in FLAP were associated with either adenoma or rectal cancer risk. Individuals with the rs12429692 (2439 A>T) homozygous variant genotype had a two-fold higher risk of adenoma (ORhzv v. wt=2.05; 95% CI 1.20-3.53; global p=0.01). Individuals who carried a variant rs17239025 (30185G>C) allele had a borderline significant 43% increased risk of rectal cancer (ORhet/hzv v. wt=1.43; 95% CI 1.01-2.04; global p=0.05). A similar pattern of higher risk in individuals with the variant allele was seen in the colon and adenoma cancer studies, though results were neither statistically significant nor noteworthy (Table 2).

There were four SNP-NSAID interactions at global p≤0.05 in the rectal cancer study (Table 3). Regular NSAID use was associated with a decreased risk of rectal cancer for individuals with the homozygous wildtype alleles of all five SNPs, as would be predicted. For three of these SNPs (rs9508832 (4527 G>A); rs9315053 (29380 T>G); and rs4075692 (113604 G>A)), regular NSAID users with a variant genotype had an additional reduction in rectal cancer risk compared to wildtype individuals (Table 3); this pattern was not seen in non-regular NSAID users.

Additionally, regular NSAID use was associated with a lower risk of colon cancer in wildtype individuals for rs17239025 (30185 G>C), whereas the protective effect conferred by NSAID use was absent among individuals with variant genotypes (p-interaction=0.02) (Table 3). The same loss of protection from NSAIDs in individuals with the variant allele was seen in both rectal cancer and adenoma, though these results were not statistically significant.

Genetic Variation in ALOX12

We observed a single significant association among the ALOX12 tagSNPs investigated. Individuals with the homozygous variant genotype of rs2073438 (639 G>A) had a 34% lower rectal cancer risk (ORhzv v. wt=0.66; 95% CI 0.42-1.04, global p=0.02) (Table 2). This association was not seen in the colon cancer or adenoma populations and there were no significant NSAID interactions with ALOX12 polymorphisms.

Genetic Variation in ALOX15

We observed evidence for associations between polymorphisms in ALOX15 and rectal cancer. Individuals with at least one variant allele of rs4796535 (1351G>A) had a 43% higher risk of rectal cancer (ORhet/hzv v. wt=1.43, 95% CI: 1.03-1.97, global p=0.03, noteworthy at 25% FDR). The association between rs2619112 (9562 C>T) and rectal cancer risk was also noteworthy at 25% FDR (ORhet v. wt=1.37, 95% CI 1.06-1.77) (ORhzv v. wt=1.13, 95% CI 0.83-1.55, global p=0.05).

We observed a significant and noteworthy NSAID interaction with rs2619112 in rectal cancer that followed the same non-dose-dependent pattern seen in the main effect analysis of this SNP: there was no protective effect for regular NSAID users with heterozygous genotypes, whereas those with homozygous wildtype and homozygous variant genotypes had lower risks (p-interaction=0.01, noteworthy at 25% FDR) (Table 3). Additionally, the lowered risk of adenoma in non-regular NSAID users with the homozygous variant genotype of rs2664593 (−189 G>C) (ORhzv v. wt=0.19, 95% CI 0.06-0.57, global p=0.04) was lost in regular NSAID users with the same homozygous variant genotype.

DISCUSSION

We combined a tagSNP and candidate-polymorphism approach to analyze genetic variation in four leukotriene pathway genes that had previously been implicated in colorectal carcinogenesis. Genetic variability in ALOX genes was most prominently associated with rectal cancer risk; although the precise pathogenic mechanisms differentially influencing colon and rectal cancer are still unclear, many studies have proposed the idea that colon and rectal cancer are distinct entities (Frattini et al., 2004; Li and Lai, 2009) in which risk is modulated by different environmental, physiologic, and etiologic mechanisms, including genetics (Caan et al., 1998; Slattery et al., 2003a, 2003b, 2004a, 2011; Haug et al., 2012). In addition, a recent study revealed differential associations between colon and rectal cancer in multiple colorectal cancer-associated loci initially identified by GWAS (Lubbe et al., 2012). Our results support these findings by indicating a differential influence of genetic variability in lipoxygenase enzymes across colorectal neoplasias, with particular importance for rectal cancer. Although these differences could stem from chance findings, the fact that many associations were observed specifically in rectal cancer suggests a true etiologic difference between colon cancer and rectal cancer.

We also observed an increased risk of adenoma associated with a FLAP polymorphism, and an NSAID interaction for an ALOX15 polymorphism in the adenoma study population. However, while adenoma may be a precursor to cancer in some individuals, in many cases adenomatous polyps never progress to cancer. Because of some heterogeneity of adenoma case outcomes, small sample sizes for certain alleles (resulting in less statistical power), and our inability to fine-tune polyp diagnoses any further than hyperplastic vs. adenomatous, this data does not provide support for etiological differences between the two cancers and adenoma.

At the time the data were collected for this study, extensive sequencing of ALOX5 had not occurred and little information regarding genetic variability was available in existing databases. Now there is evidence for much more variability in ALOX5, including another promoter SNP (−1753 G>A) that is in complete LD with the functional candidate SNP in our study rs4986832 (−1700G>A), and which has been associated with a reduced risk of colon cancer (Goodman et al., 2004). We also observed a trend for lower risk of cancer for individuals with the variant genotype rs4986832, specifically in the rectal cancer population.

The variant genotypes of the ALOX5 VNTR were also statistically significantly associated with a decreased risk of rectal cancer, with a similar, although statistically non-significant, association in the colon cancer population. Variability in the ALOX5 VNTR has been associated with transcription modification in vitro (In et al., 1997; Silverman and Drazen, 2000; Wittwer et al., 2006), and downstream eicosanoid production has been shown to be higher in individuals with the wildtype 5/5 genotype than for individuals with at least one allele containing fewer than five repeats (Stephensen et al., 2011). Additionally, this polymorphism has been implicated in inflammation-associated diseases such as asthma (Kalayci et al., 2006) and atherosclerosis (Dwyer et al., 2004), and we previously observed an interaction between this polymorphism and a functional promoter polymorphism in COX-2 in the adenoma case-control study (Poole et al., 2006). Taken together, these findings suggest an important role for the ALOX5 VNTR in the pathogenesis of inflammatory diseases.

The functional candidate polymorphisms included in our study have not, to our knowledge, been included in a GWAS for colorectal cancer risk. However, even if they had been added to a GWAS SNP panel, pooling of colon and rectal cancer in the study populations may have attenuated the ability to detect associations. In addition, the relative technical difficulty of genotyping VNTRs has limited their inclusion in other studies, though advances in next generation sequencing will allow for easier sequencing of repeat polymorphisms (Schentrup et al., 2009). Further studies are needed to investigate these and more recently discovered polymorphisms in ALOX5 and their relation to colorectal neoplasia.

FLAP, the 5-lipoxygenase-activing protein, partners with ALOX5 to regulate leukotriene pathway activity. Four polymorphisms in FLAP showed statistically significant interactions with NSAID use and rectal cancer. Though none of the associations were noteworthy at 25% FDR, the consistent patterns of risk and repeated findings in the rectal population are intriguing. Although FLAP polymorphisms have not previously been assessed in the context of colorectal neoplasia, genetic variation in FLAP has been associated with increased risk of a variety of inflammatory diseases including stroke, asthma, and cardiovascular disease (Domingues-Montanari et al., 2010; Huang et al., 2010; Via et al., 2010), though some analyses yielded conflicting results (Matarin et al., 2009). It is likely that the result of genetic variation in FLAP is too subtle to be detected in most single-gene SNP analyses; rather, there may be gene-gene interactions occurring within the 5-lipoxygenase pathway, such as those detected in asthma between FLAP and downstream leukotriene LTA4H (Holloway et al., 2008; Via et al., 2010). As FLAP is the activating protein for ALOX5, FLAP-ALOX5 interactions seem likely. Though we were not able to properly assess a gene-gene interaction using only candidate polymorphisms in ALOX5, we did not detect interactions between individual ALOX5 and FLAP polymorphisms in a preliminary analysis. Potential epistatic effects remain an important avenue of research. Overall, genetic variation in FLAP could provide new insights into the lipoxygenase pathway and colorectal carcinogenesis, especially if the focus is on rectal cancer and NSAID use.

The downstream product of ALOX12, 12-HETE, can enhance carcinogenesis by stimulating cell proliferation, angiogenesis, and tumor spread, and inhibiting apoptosis (Chopra et al., 1991; Tang et al., 1996). These findings have generated research interest in ALOX12 polymorphisms in a variety of diseases, since altered enzyme expression caused by genetic variation could have a direct influence on cancer risk. R261Q, the single most extensively studied ALOX12 polymorphism in relation to colorectal neoplasia risk, was previously observed to be associated with a 40% lower colorectal adenoma risk and had a significant NSAID interaction in an adenomatous polyp study (Gong et al., 2007). Conversely, the same polymorphism was associated with higher risk of both rectal and colon cancer in another study (Tan et al., 2007); and yet another study reported no associations between this SNP and risk of colon cancer (Goodman et al., 2004). We did not observe a statistically significant association with cancer risk in the included tagSNP that is in perfect LD with (and has a similar MAF to) R261Q (rs434473 N322G, r2=1). However, the Gong, et al. and the Goodman, et al. studies had substantially smaller sample sizes than our study (N=162 cases and 178 cases respectively), which can lead to false positives and inconsistencies; additionally, the Tan, et al. study, which had more robust sample sizes (N=403 colon cancer and 597 rectal cancer cases) was focused on a Chinese Han population. It is possible that there are different modifying alleles or health behaviors in these different ethnic populations. The one ALOX12 SNP in our study that was statistically significantly associated with a 44% lower rectal cancer risk (rs2073438) is in moderate LD (r2 ~ 0.5) with these two functional ALOX12 polymorphisms, though this probably represents an independent effect. Overall, in light of the inconsistency in results among the previous studies in different populations, studies of interactions with modifying alleles or health behaviors in specific populations are warranted.

The ALOX15 SNP rs2619112 (9562G>A) emerged as an interesting variant in our study, with a borderline significant increased risk of rectal cancer in individuals carrying the heterozygous AG genotype, and a statistically significant and noteworthy NSAID interaction. Similar patterns of elevated risk for disease in individuals with the heterozygous genotype of this polymorphism have been observed in Chinese Han populations with coronary artery disease (Zhang et al., 2010). Furthermore, interactions between rs2619112 and menopausal status modified risk of low bone mineral density in women (Cheung et al., 2008). One additional ALOX15 SNP (rs4796535, 1351G>A) was associated with an increased risk of rectal cancer, also at 25% FDR. Previous studies have shown that down-regulation of ALOX15 can lead to colorectal carcinogenesis, warranting further study of this pathway as a potential therapeutic target (Bhattacharya et al., 2009).

Our study design has several strengths and limitations. We were able to investigate more rare and functional polymorphisms not covered in current GWAS. Use of identical genotyping platforms across three independent case-control studies enabled us to investigate the full continuum of genetic variation across colorectal carcinogenesis. Sample sizes were relatively large, though stratification by NSAID use was sometimes underpowered, and this study was not powered to capture the impact of rarer polymorphisms or associations in multiple ethnicities. Few associations were noteworthy at 25% FDR, and none would have been significant after very stringent methods of correction for multiple comparisons. However, if we view these results as avenues toward potentially fruitful areas of future research rather than definitive or clinically actionable findings, discussion of uncorrected results with p≤0.05 can be considered valid, though we acknowledge that we are more open to type I errors when we minimize type II errors (Streiner and Norman, 2011). Also, assessing the genetic variation in ALOX5 and ALOX15 was limited due to the lack of resequencing of the ALOX5 gene at the time of the study, and general difficulties in genotyping this gene due to the presence of an ALOX15 pseudogene.

Our results suggest that genetic variability in the lipoxygenase pathway impacts the risk of colorectal neoplasia, and that variability in FLAP and ALOX15, in particular, may modify the protective association of NSAID use against colorectal neoplasia, especially rectal cancer. NSAIDs are emerging as potent cancer-preventive agents, yet have a risk of adverse effects (Ulrich et al., 2006; Cross et al., 2008; Chan et al., 2012); tailoring NSAIDs to individuals rather than prescribing them indiscriminately is a key goal of pharmacogenetics. These and other studies examining genetic variability, risk of colorectal cancer, and NSAID use interactions provide valuable insight into disease etiology, future therapeutic targets, and the potential for individualized treatment.

Supplementary Material

Supp Table S1-S3

Table 3A.

Interactions between lipoxygenase tagSNPs, NSAID use and risk of colorectal neoplasia

Colon Cancer NSAID Use
Rectal Cancer NSAID Use
Adenoma NSAID Use
Non-Regular Regular Non-Regular Regular Non-Regular Regular



SNP name Geno-
type
Case/
Control
OR
(95% CI)
Case/
Control
OR
(95% CI)
pintb Case/
Control
OR
(95% CI)
Case/
Control
OR
(95% CI)
pintb Case/
Control
OR
(95% CI)
Case/
Control
OR
(95% CI)
pintb
FLAP

rs17239025 GG 845/920 1.00 409/684 0.60 311/371 1.00 199/322 0.67 282/299 1.00 149/229 0.61
30185 G>C (referent) (0.51-0.71) (referent) (0.53-0.85) (referent) (0.50-0.80)
GC/CCa 94/104 0.96 57/58 1.03 0.02 48/41 1.38 22/27 1.06 0.73 26/25 1.23 25/29 0.84 0.82
(0.71-1.29) (0.70-1.52) (0.88-2.16) (0.58-1.93) (0.6-2.4) (0.50-1.60)

rs9508832 GG 299/306 1.00 144/238 0.58 121/132 1.00 70/111 0.64 103/87 1.00 65/87 0.55
4527 G>A (referent) (0.44-0.76) (referent) (0.43-0.95) (referent) (0.34-0.89)
AG 453/501 0.95 237/371 0.62 165/209 0.88 121/170 0.76 144/171 0.68 81/126 0.46
(0.77-1.17) (0.49-0.79) (0.64-1.22) (0.54-1.08) (0.45-1.02) (0.30-0.73)
AA 189/213 0.92 86/138 0.61 0.72 73/70 1.10 29/77 0.38 0.02 62/65 0.82 28/45 0.45 0.76
(0.71-1.18) (0.44-0.84) (0.72-1.67) (0.23-0.63) (0.49-1.35) (0.24-0.84)

rs9315053 TT 412/444 1.00 217/343 0.64 157/191 1.00 107/159 0.77 154/134 1.00 93/117 0.61
29380 T>G (referent) (0.51-0.80) (referent) (0.55-1.07) (referent) (0.41-0.91)
GT 419/449 1.03 215/328 0.67 155/184 1.00 98/157 0.73 124/152 0.73 69/115 0.45
(0.85-1.24) (0.53-0.84) (0.74-1.36) (0.52-1.02) (0.50-1.05) (0.30-0.69)
GG 105/125 0.95 34/73 0.49 0.68 47/36 1.58 16/42 0.44 0.03 31/38 0.75 11/26 0.45 1.00
(0.71-1.28) (0.32-0.76) (0.97-2.59) (0.23-0.81) (0.42-1.34) (0.20-1.02)

rs4075692 GG 240/256 1.00 129/200 0.64 101/108 1.00 114/214 0.61 95/77 1.00 59/67 0.63
13604 G>A (referent) (0.48-0.85) (referent) (0.39-0.94) (referent) (0.37-1.05)
AG 489/528 1.00 237/377 0.64 177/220 0.85 93/130 0.75 140/168 0.66 88/133 0.46
(0.80-1.24) (0.50-0.82) (0.60-1.20) (0.52-1.08) (0.43-1.00) (0.29-0.72)
AA 212/236 0.99 99/168 0.61) 0.97 81/84 0.97 14/15 0.40 0.03 74/78 0.82 27/58 0.37 0.50
(0.77-1.29) (0.44-0.83 (0.64-1.47) (0.25-0.64) (0.50-1.33) (0.20-0.67)

rs9551960 AA 329/352 1.00 149/263 0.57 127/125 1.00 69/117 0.53 98/104 1.00 53/81 0.75
7202 G>A (referent) (0.44-0.74) (referent) (0.36-0.79) (referent) (0.45-1.23)
AG 448/491 0.99 241/341 0.71 167/198 0.81 106/189 0.51 157/166 1.20 84/129 0.65
(0.81-1.21) (0.56-0.89) (0.58-1.12) (0.36-0.73) (0.81-1.78) (0.42-1.01)
GG 161/179 0.95 76/141 0.55 0.35 64/87 0.69 45/53 0.82 0.05 54/52 1.21 36/184 0.73 0.61
(0.73-1.24) (0.40-0.76) (0.46-1.05) (0.51-1.32) (0.72-2.03) (0.42-1.29)

ALOX15

rs2619112 CC 278/301 1.00 127/210 0.61 81/123 1.00 48/91 0.58 83/94 1.00 53/70 0.70
9562 C>T (referent) (0.46-0.81) (referent) (0.37-0.90) (referent) (0.42-1.17)
CT 470/501 1.01 245/362 0.68) 174/198 1.10 125/164 1.00 157/166 0.97 87/129 0.65
(0.82-1.25) (0.54-0.86 (0.79-1.55) (0.69-1.43) (0.64-1.46) (0.42-1.02)
TT 190/216 0.93 93/174 0.53 0.71 98/88 1.30 46/96 0.49 0.01 69/64 1.30 33/59 0.55 0.38
(0.71-1.20) (0.39-0.73) (0.86-1.97) (0.31-0.80) (0.78-2.17) (0.31-0.98)

rs2664593 CC 561/654 1.00 288/460 0.68 215/255 1.00 0.75 190/185 1.00 110/174 0.55
−189 G>C (referent) (0.56-0.82) (referent) (0.57-1.00) (referent) (0.38-0.78)
CG 320/317 1.14 146/246 0.64 130/137 1.07 70/129 0.61 114/120 0.95 59/78 0.61
(0.94-1.39) (0.50-0.81) (0.79-1.45) (0.43-0.86) (0.66-1.37) (0.39-0.96)
GG 55/46 1.31 33/36 0.95 0.48 14/20 0.93 5/14 0.39 0.38 5/19 0.19 5/6 0.93 0.04
(0.86-1.99) (0.58-1.56) (0.45-1.92) (0.14-1.14) (0.06-0.57) (0.26-3.28)
a

the p-interaction for the dominant model is provided when any cell < 5

b

Italics underlined = noteworthy at 25% FDR

ACKNOWLEDGMENTS

The authors would like to thank Dr. Roberd Bostick and Lisa Fosdick for their contributions to the initial establishment of the adenoma study, Dr. Kristin Anderson, Sandie Edwards, and Donna Morse for their contributions to the colon and rectal cancer studies.

Supported by: National Cancer Institute grants R01 CA114467, R03 CA123577, R25 CA094880, CA48998, and T32 CA009001

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