COX-2 gene promoter haplotypes and prostate cancer risk
Ramesh C.K. Panguluri1,4,
Layron O. Long1,3,
Weidong Chen1,
Songping Wang1,
Aoua Coulibaly1,
Flora Ukoli3,
Aaron Jackson2,
Sally Weinrich5,
Chiledum Ahaghotu1,3,
William Isaacs6 and
Rick A. Kittles1,3,7
1 National Human Genome Center at Howard University, 2 Divison of Urology, Howard University Hospital and 3 Howard University Cancer Center, Washington, DC 20060, USA, 4 National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arizona, USA, 5 School of Nursing, University of Louisville, Louisville, KY 40292, USA and 6 Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA
7 To whom correspondence should be addressed Email: rkittles{at}howard.edu
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Abstract
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Cyclooxygenase-2 (COX-2) is a key rate-limiting enzyme that converts arachidonic acid into pro-inflamatory prostaglandins. COX-2 expression is strongly correlated with increased tumor microvasculature density and plays an important role in inhibiting apoptosis, stimulating angiogenesis and promoting tumor cell metastasis and invasion. However, little is known about the role that sequence variation of the COX-2 gene contributes to prostate cancer. Thus, we searched for polymorphisms in the promoter region of the COX-2 gene using denaturing high-performance liquid chromatography. Four single nucleotide polymorphisms (SNPs), 1285A/G, 1265G/A, 899G/C and 297C/G, were detected and confirmed by direct sequencing. Three of the SNPs in the promoter region of COX-2 gene create at least three putative transcription factor binding sites and eliminate CCAAT/enhancer binding protein alpha (C/EBP
) and NF-
B binding sites. A case-control study of the four SNPs in African American (n = 288), Bini Nigerian (n = 264) and European American (n = 184) prostate cancer cases and age-matched controls revealed that SNP 297G was associated with a decreased risk for prostate cancer [odds ratio (OR) = 0.49; CI = 0.20.9; P = 0.01]. The effect on risk was observed in both African Americans (OR = 0.51; CI = 0.20.9; P = 0.01) and European Americans (OR = 0.33; CI = 0.10.9; P = 0.02). In addition, SNPs 1265A and 899C were associated with increased prostate cancer risk in African Americans (OR = 2.72; CI = 1.35.8; P = 0.007 and OR = 3.67; CI = 1.49.9; P = 0.007, respectively). Haplotype analyses revealed modest effects on susceptibility to prostate cancer across populations. Haplotype GGCC conferred increased risk in the African American and Nigerian populations. Conversely, haplotype AGGG exhibited a negative association with prostate cancer risk in African Americans (OR = 0.4; CI = 0.10.9; P = 0.02) and European Americans (OR = 0.2; CI = 0.10.9; P = 0.03). These data suggest that variation of the COX-2 promoter may influence the risk and development of prostate cancer.
Abbreviations: C/EBP
, CCAAT/enhancer binding protein alpha; COX, cyclooxygenase; DRE, digital rectal exam; PSA, prostate specific antigen; SNPs, single nucleotide polymorphisms; TNM, tumor node metastasis
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Introduction
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Prostaglandins play a key role in cellular proliferation and growth of both normal and aberrant cells. Furthermore, prostaglandins mediate the effects of several growth promoting factors and oncogenes (1,2). Two isoforms of cyclooxygenase (COX-1 and COX-2) are known rate-limiting enzymes involved in the production of prostaglandins from arachidonic acid precursors (3). The constitutive cyclooxygenase (COX-1) is present in many tissues and synthesizes prostaglandins that are involved in maintaining normal tissue homeostasis (4). The second isoform, COX-2, is responsible for prostaglandins produced in sites of inflammation and is induced by growth factors, cytokines and various carcinogens (5). Most importantly, COX-2 is also involved in mechanisms of carcinogenesis such as apoptosis (6,7), invasiveness (8), adhesion (9) and angiogenesis (10). Several studies have indicated that COX-2 is up-regulated in various cancers: breast, colon, lung, pancreas, esophagus and prostate (1117).
The promoter region of the COX-2 gene consists of various transcriptional regulatory elements, which may have profound effects on expression of the enzyme. Major upstream transcriptional regulatory elements identified previously in the promoter include: a scaffold/matrix attachment region, CCAAT/enhancer binding protein (C/EBP) site, AP1-like site, GAS-binding site, SIE-binding, NF-
B, AP2-like site and a TBP-binding site (18).
To elucidate the role of the COX-2 gene in prostate cancer development and progression, we examined DNA sequence variation in the COX-2 promoter in several clinically evaluated populations from three different ethnic groups. In order to better determine the role sequence variation plays in prostate cancer etiology it is important to examine diverse populations because of potential genetic and environmental differences among populations. These differences may contribute to the incidence of prostate cancer varying significantly across ethnic groups with African American men having the highest incidence of prostate cancer compared with other ethnic groups. In addition, age-adjusted mortality is two times higher for African American men than for European American men (19).
In this study we identified single nucleotide polymorphisms (SNPs) in the COX-2 gene promoter and performed genotype and haplotype association analyses on prostate cancer in African Americans, Nigerians and European Americans. We observed strong but different patterns of associations between COX-2 SNP genotypes, haplotypes and prostate cancer among the three ethnic groups. Our results provide evidence that sequence variation in the COX-2 promoter contributes to risk of prostate cancer.
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Materials and methods
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Subjects
Unrelated men from three different ethnic groups were enrolled for case-control studies of risk factors for prostate cancer. All prostate cancer cases were between 40 and 85 years of age and were diagnosed with prostate cancer within the last 4 years. The first group consisted of 288 African Americans (124 prostate cancer patients and 164 healthy male controls) recruited from the Howard University Hospital (HUH) in Washington, DC. Incident cases were identified through the Division of Urology at HUH and confirmed by review of medical records. The participation response rate among the African American cases was 92%. Healthy control subjects matched for age were ascertained from the Division of Urology at HUH and also from men participating in screening programs for prostate cancer at the HUH. The screening program was demographically similar to the patient population seen in the Division of Urology clinics. The recruitment of controls occurred concurrently with individuals recruited with prostate cancer. The participation response rate for the African American controls was 90%.
The second group of prostate cancer cases and controls consisted of 184 European Americans (92 prostate cancer patients and 92 healthy male volunteers) recruited from the Johns Hopkins Hospital (Baltimore, MD) (21). The European American prostate cancer case subjects were recruited from the patients who underwent prostate cancer treatment at Johns Hopkins Hospital. The diagnosis of prostate cancer was confirmed by pathology reports. The response rate among the European American cases was 95%. European American control subjects were recruited from volunteers participating in prostate cancer screening programs at Johns Hopkins Hospital. The response rate for the European American controls was over 90%.
A third group of prostate cancer cases and controls consisting of 264 Nigerians (Edo State) (154 prostate cancer patients and 110 healthy male controls) belonging to the Bini ethnic group were enrolled through a community-based study of risk factors for prostate cancer in collaboration with the University of Benin Teaching Hospital (UBTH) in Benin City, Nigeria (20). Incident cases were recruited during the years 2000 and 2001 at UBTH. During the 2-year period healthy controls were also ascertained from a prostate cancer screening program in the same community as UBTH. The Bini ethnic group represents 84.7% of the population in the Benin City community. The response rate among the Nigerian cases and controls were 88 and 85%, respectively. Blood samples were collected from each subject. Clinical characteristics including Gleason grade, prostate specific antigen (PSA), tumor node metastasis (TNM) stage, age at diagnosis and family history were obtained from medical records. Disease aggressiveness was defined as Low (T category <T2c and/or Gleason grade <7) or High (T category
T2c and/or Gleason grade
7). All healthy controls had PSA levels <4.0 ng/ml and normal digital rectal exam (DRE). The Howard University Institutional Review Board approved the study and written consent was obtained from all subjects.
SNP discovery
Genomic DNA was isolated from lymphocytes by standard proteinase K digestion, cell lysis, protein precipitation and DNA precipitation. 1400 bp upstream of the ATG start site of the COX-2 gene was screened for DNA sequence variation by denaturing high-performance liquid chromatography using the WaveTM DNA fragment analysis system (Transgenomic, Omaha, NE) according to manufacturer's instructions. Five sets of primers used to amplify COX-2 promoter fragments for SNP discovery were AM1F 5'-GCT GTC AAA ATC TCC CTT CC-3' and AM1R 5'-TTT CTC TCC CTG ATG CGT GG-3'; AM2F 5-AGT AAC CAA AAT AAT CCA CGC-3' and AM2R 5'-TAC CTT CAC CCC CTC CTT G-3'; AM3F 5'-TTA CCT TTC CCG CCT CTC-3' and AM3R 5'-GTT TTA TGT TTT AGT GAC GAC GC-3'; AM4F 5'-GCT ATG TAT GTA TGT GCT GC-3' and AM4R 5'-GCT TCC GAG AGC CAG TTC-3'; AM5F 5'-CGG TAT CCC ATC CAA GGC-3' and AM5R 5'-TGC TCC TGA CGC TCA CTG-3'. Genomic DNA from 20 individuals (10 prostate cancer cases and 10 controls) from each of the three populations (total 60) were used for SNP discovery. We detected four polymorphisms, 1285A/G, 1265G/A, 899G/C and 297C/G (dbSNP # ss5112604, ss5112605, ss5112606 and ss5112607, respectively).
Genotyping
The 1265G/A SNP was genotyped by direct DNA sequencing from the PCR product using fluorescent labeled Big Dye sequencing chemistry (ABI) and the ABI 377 DNA sequencer (Foster City, CA). Genotyping of the 1285A/G, 899G/C and 297C/G polymorphisms was performed using PyrosequencingTM (Pyrosequencing, AB, Uppsala, Sweden) according to standard protocols (22). PCR amplification of the 1285A/G SNP used a forward primer, 5'-TTC CAG CTG TCA AAA TCT C-3' and a biotinylated reverse primer, 5'-biotin-AAG ATT ATG AGT TGT GAC C-3'; for the 1265G/A SNP, forward primer 5'-ATC TCC CTT CCA TCT AAT-3' and reverse primer 5'-TGG TAA AAA TAA ATT CGA GT-3'; for the 899G/C SNP, forward primer 5'-ACA GGG TAA CTG CTT AGG-3' and biotinylated reverse primer 5'-biotin-CTC CTT GTT TCT TGG AA-3'; and for the 297C/G SNP, forward primer 5'-AGA CAG GAG AGT GGG GAC-3' and reverse biotinylated primer 5'-biotin-GGG GGC AGG GTT TTT TAC-3'. Pyrosequencing primers were as follows: 5'-CCA AAA CGA GAA TA-3' for 1285A/G; 5'-GAG GAG AAT TTA C-3' for 899G/C; and 5'-TTC CGA TTT TCT CAT T-3' for 297C/G. Genotyping of these three SNPs were performed using the PSQ96 automated Pyrosequencing instrument (Pyrosequencing AB). All samples were genotyped twice directly from genomic DNA. Control DNAs included known wild-type homozygotes, heterozygotes and variant homozygote sample genotypes. The control DNAs were confirmed by direct DNA sequencing using an ABI 377 DNA sequencer (ABI). Genotypes from the repeat assay were 100% concordant with initial genotypes.
Haplotype construction and statistical analyses
Genotype and allele frequencies were calculated for each population. HardyWeinberg equilibrium analysis of each group was evaluated by contingency table analysis. COX-2 haplotypes were estimated from the marker genotypes by a maximum likelihood method using the computer program MLOCUS (written by J.Long), which implements the EM algorithm (23,24). The MLOCUS program was also used to calculate pairwise linkage disequilibrium (D') for each marker pair. Odds ratios and P values were determined by multiple logistic regression analyses from comparison of genotypes or haplotypes between prostate cancer patients and healthy controls using SAS version 6.12 (SAS Institute, Cary, NC). Analyses were performed initially on the combined dataset consisting of all three populations. For all analyses of the combined dataset, genetic effects were adjusted for the potential confounders such as ethnicity, age (at time of diagnosis for case subjects and at time of ascertainment for controls) and family history (defined as one or more first degree relatives). Since there may be specific genetic effects within different populations (environments), analyses were also performed for individual ethnic groups.
Electronic database information
The Alibaba2 web-based program was used to predict transcription factor binding sites in the promoter sequence of the COX-2 gene http://gene-regulation.com; dbSNP for SNP information http://www.ncbi.nlm.nih.gov/dbSNP; Online Inheritance in Man (OMIM) http://www.ncbi.nih.gov/OMIM/ [for COX-2 (MIM 600262) and RNASEL (MIM 180435)].
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Results
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Table I describes the patient populations. Mean ages for cases and controls differed across, but not within, the three ethnicity groups. There were no significant differences in the distribution of PSA levels between African Americans and European Americans; however, subtle differences were evident between African Americans and European Americans for mean age at diagnosis and family history of prostate cancer. The percentage of men with family history for prostate cancer was much higher for African Americans. Nigerian subjects were much older, exhibiting more aggressive disease and much higher PSA than their US counterparts.
Four SNPs were identified in the COX-2 gene promoter (1285A/G, 1265 G/A, 899 G/C and 297 C/G). The 899C SNP was reported previously in a genetic study on familial adenomatous polyposis (25). Table II shows allele frequencies of the four polymorphisms in the three normal control populations. Significant differences in allele frequencies (P < 0.01) were observed for the 1285G, 1265A and 899C SNPs between African Americans and the other two populations (Table II). Putative changes in transcription factor binding sites due to these SNPs were evaluated by computer prediction using the Alibaba2 program (26). Table III shows the various transcription factor binding sites that were created or lost due to the four polymorphisms in the COX-2 gene promoter.
Genotype frequencies are shown in Table IV. All genotypes were in HardyWeinberg equilibrium for each population (P > 0.05; data not shown). Allele and genotype frequencies for African Americans were intermediate between Bini Nigerians and European Americans for all four SNPs. Analyses of the markers in the combined dataset controlling for ethnicity, age at diagnosis and family history revealed that the presence of the 297G allele decreased risk for prostate cancer [odds ratio (OR) = 0.49; 95% CI = 0.20.9]. Subset analyses of the different ethnic groups revealed the effects of 297 CG/GG genotypes on prostate cancer risk in African Americans (OR = 0.51; 95% CI = 0.20.9; P = 0.01) and similarly among European Americans (OR = 0.33; 95% CI = 0.080.89; P = 0.02).
Analyses of the other three SNPs in the combined dataset did not indicate an association with prostate cancer (Table V). However, in subset analyses with the African American population, 1265 GA/AA genotypes were associated with increased prostate cancer risk (OR = 2.72; 95% CI = 1.35.8; P = 0.007). In addition, individuals with at least one copy of the 899C allele were at increased risk for prostate cancer in the African American population (OR = 3.67; 95% CI = 1.49.9; P = 0.007) (Table V). As a result of the low frequency of the 1265A and 899C SNPs in the European American population there lacked sufficient power to determine an association with the disease.
Altogether six haplotypes comprise >97% of all chromosomes observed in the African American and Nigerian populations (Table IV). However, only two haplotypes accounted for 99% of the European American chromosomes. Pairwise linkage disequilibrium (LD) analyses revealed moderate to strong LD across marker pairs for all populations. No significant differences in pairwise LD patterns existed between prostate cancer cases and controls. Haplotype AGGC was the most common haplotype among all populations ranging from 52 to 93% in Nigerian and European American control populations, respectively.
Haplotype AGGG was significantly more common among control subjects (8%) than cases (4%) in the combined dataset (Table IV). This pattern was observed across all three ethnic groups. Table VI shows that even after controlling for ethnicity, age and family history of prostate cancer, haplotype effects were observed in the combined dataset for haplotypes AGGG (OR = 0.4; CI = 0.20.7; P = 0.002) and GGCC (OR = 1.4; CI = 1.12.2; P = 0.04). These effects were also observed in the subset analyses of the ethnic groups. Specifically, haplotype AGGG was negatively associated with prostate cancer in African Americans and European Americans (OR = 0.4; CI = 0.10.9; P = 0.02 and OR = 0.2; CI = 0.10.9; P = 0.03, respectively). Among African Americans, the frequency of haplotype AGGG was 3% for affected men and 9% for controls. Likewise, the frequency for haplotype AGGG among European Americans was 2% for affected men and 6% for controls. Among the Bini Nigerians with haplotype AGGG, the trend was similar (6% of cases and 9% of controls), yet not significant (P = 0.58).
Subset analyses also revealed that haplotype GGCC was strongly associated with prostate cancer in African Americans (OR = 4.5, CI 1.316; P = 0.01) and Nigerians (OR = 3.9, CI = 1.119; P = 0.003). Additionally, African Americans possessing haplotype AACG exhibited increased risk for prostate cancer (OR = 2.5; CI = 1.25.1; P = 0.006). Analyses in which we stratified all prostate cancer cases according to disease aggressiveness, age at diagnosis and family history revealed no significant correlations with individual SNPs, haplotypes and the disease (data not shown).
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Discussion
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Numerous studies suggest a role for COX-2 in carcinogenesis (69,11,14). However, little is known about the role sequence variation within the COX-2 gene plays in prostate cancer risk. This is the first genetic study that explores the impact DNA sequence variation within the COX-2 promoter has in prostate cancer risk in diverse ethnic groups. In this study, we identified four SNPs within a 1.4 kb region upstream of the COX-2 gene ATG-site. We utilized SNPs and haplotypes for a case-control association study on prostate cancer risk. Since the four SNPs were in close proximity (<1 kb) we observed strong linkage disequilibrium in the populations. Thus, the four SNPs were not independent of each other and probably carry similar information about risk. By evaluating the single markers collectively as haplotypes, the SNP associations were confirmed. However, our results should be viewed with caution since association studies using unrelated controls might be affected by population stratification. Population stratification occurs when residual confounding by ethnicity remains due to unidentified ethnic differences between cases and controls. However, it is unlikely that population stratification caused confounding in this study since an unlinked marker analysis consisting of 20 SNPs with large differences in allele frequencies between west African and European ancestry within three populations revealed no significant differences between cases and controls (R.A.K., W.C. and L.L., unpublished observations).
Patterns of COX-2 haplotype variation differed between populations. Populations of African descent possessed more than twice the number of haplotype observed among European Americans. This observation of higher haplotype variation among African descent populations is consistent with studies that focus on variation at other genetic markers (21,27). Greater genetic diversity in African populations may have broad implications on our understanding of genetic diseases and the common disease/common variant hypothesis (2830). The presence of the same haplotypes conferring risk in different populations provides intriguing evidence for the involvement of COX-2 in prostate cancer risk. However, this study also revealed that different COX-2 haplotypes were implicated in prostate cancer risk in the different populations. For instance, for African Americans and Bini Nigerians, the GGCC haplotype increased the risk for prostate cancer, while in African Americans and European Americans, a different haplotype, AGGG, probably contributes protective effects. It is possible that other causative genetic variants may lie either upstream or downstream of the SNP markers found in our study. In addition we note that there may be other limitations in this study. Besides small sample sizes in the subset analyses, the functional significance of these sequence polymorphisms is lacking.
Recently, the prostate cancer susceptible gene, 2'-5'-oligoadenylate (2-5A)-dependent RNase L (RNASEL) was mapped by Carpten et al. (31) [OMIM 180435] to the HPC1 locus on the chromosome 1 (1q24-25). Interestingly, the COX-2 gene is also mapped to the same region on chromosome 1 (1q25.2). Since COX-2 is located close to HPC1 and RNASEL was associated recently with sporadic disease (32), we also typed several common RNASEL SNPs (95T/C, 1385G/A and 1623T/G) in our populations and did not observe any associations. Thus, it would be of interest to explore the involvement of COX-2 gene in HPC.
DNA sequences upstream of the COX-2 translation initiation site possess multiple putative transcription factor binding sites. Of the SNPs observed in the study populations, two (1265A and 297G) lead to the creation of two new transcription factor binding sites while one polymorphism (899C) eliminates a transcription factor-binding site (see Table II). The 1265A SNP eliminates a C/EBP
binding site on the COX-2 promoter and creates Pit-1a and Hb binding sites. C/EBP
regulates cell proliferation and gene expression (3335). In vivo studies on mice reveal that the loss of C/EBP
results in a 2055% decrease in the transcription of COX-2 gene (36). The 899C SNP is within a Sp-1 binding site, but does not appear to change the consensus binding site, however, a NF-
B site is eliminated upstream of the SP-1 binding site. Multiple studies have shown that COX-2 is over expressed in prostate cancer cells (15,37), and that the induction of COX-2 expression is mediated by NF-
B (38).
Our study reveals the complexity of multiple SNP analyses where different SNPs may alter transcriptional binding sites. What may be a protective SNP in one population may appear to increase risk in another population due to other linked polymorphisms, which may exhibit stronger effects on gene function. For instance, the 297G allele creates two new sites, a ribosomal DNA enhancer binding protein 1 site, and a C/EBP
site that plays an important role in cell-cycle growth arrest in mammary epithelial cells and fibroblasts (39). Thus, the creation of the new binding sites due to the 297G allele may be protective against tumorogenesis and hence be negatively associated with prostate cancer risk. If the above predicted changes in the transcription factor binding sites are valid, they could have major consequences for the expression of COX-2. Functional studies or expression studies in tumor tissues of known COX-2 haplotypes are strongly warranted in order to answer fully the various points discussed here.
The close proximity, strong LD, possible regulatory significance and the ability to construct haplotypes using COX-2 promoter SNPs increased our power to evaluate the role variation in the COX-2 gene plays in prostate cancer risk. Our results reveal a complex pattern of COX-2 haplotype associations with prostate cancer. The complex pattern of haplotype association is due mainly to polymorphisms within transcription binding sites segregating on different haplotypes. Haplotypes with the nucleotide C at the 899 position of COX-2 promoter appear to increase susceptibility and haplotypes with G at the 1285 position appear to have a protective effect. These observations clearly require further detailed investigation of the functional and expression consequences of the COX-2 promoter SNPs.
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Acknowledgments
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We thank all volunteers who participated in this study. We appreciate the assistance of K.Joseph and Drs U.Osime and F.Akenyeni in subject enrollment and data management. We also thank Dr C.Bonilla and two anonymous reviewers for helpful comments on the manuscript. This work was supported by NIH (RR03048-13S1 and 1U54CA91431-01), the Department of Defense (DAMD17-00-1-0025 and DAMD 17-02-1-0067), and the Howard University Cancer Center.
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Received September 16, 2003;
revised January 12, 2004;
accepted January 17, 2004.