In this population-based case–control study, there was evidence that COX-2 genetic variation may be modestly associated with breast cancer risk. As COX-2 plays an important role in inflammation and wound healing, it may be that an evolutionary disadvantage would be conferred to those with functional polymorphisms in this gene such that there are not large differences in function associated with genetic variation or that other factors are more important in terms of control of protein expression. We also found that there may be some interaction of at least one genetic variant with aspirin use.
Our findings were generally consistent with previous studies and extend those findings by assessing interaction with recent and adult lifetime aspirin use, and recent ibuprofen use. We observed a statistically significant increased risk of breast cancer with rs2745559. Consistent with our finding, Gallicchio et al. [11
] reported an increased risk with the variant of rs2745557, a SNP in linkage disequilibrium with rs2745559 (r2
= 1; OR 2.07, 95% CI 0.78–5.48). However, in another study, the Breast and Prostate Cancer Cohort Consortium (BPC3), a large pooled analysis of prospective studies including 6,292 breast cancer cases and 8,135 matched controls, the authors reported no association of breast cancer risk with rs2745557 [18
We found a modest inverse association with breast cancer and the COX
SNP, rs5275. This finding is consistent with that reported in a recent meta-analysis of this SNP in association with breast cancer risk (OR 0.84, 95% CI 0.70–1.00) [30
]. Results of individual studies of this SNP with breast cancer have not been statistically significant [9
]. Only in one study [13
], a hospital-based case–control study in Austria was the association of rs5275 and breast cancer risk statistically significant (OR 2.03, 95% CI 1.30–2.25); however, in that study, genotype frequencies were not in HWE among controls.
SNP, rs2143416, was inversely associated with risk in our study. In contrast, Gallichio et al. [11
] recently reported a non-significant increase in breast cancer risk with the homozygote variant of rs2143416 (OR 2.19, 0.74–6.46) in a small nested case–control study of women with benign breast disease. Others studied that a SNP in linkage disequilibrium with rs2143416, rs20417, reported no association with breast cancer [10
We observed no association of rs2206593 and breast cancer risk, similar to BPC3 [18
]. Two studies reported increases in risk (OR ≥ 2.3), but due to very small MAF in Caucasians, confidence intervals were wide [9
]. Consistent with the BPC3 [18
], we observed no association between rs689466 and breast cancer. In contrast, Gao et al. [12
] reported an increase in breast cancer risk in a hospital-based case–control study conducted in Nanjing, China (OR 1.31, 95% CI 0.95–1.81). Only the BPC3 has reported on rs12042763 and rs4648261 in association with breast cancer risk [18
]. Whereas we observed a suggestion of reduced risk among carriers of the homozygous variant of rs12042763, and no association among carriers of the rs4648261 variant allele, the authors of the BPC3 found no association with either SNPs [18
Two haplotypes that included rs2745559 minor alleles were associated with increased risk of breast cancer. Although others have investigated haplotypes with risk of prostate or breast cancer, none have included this SNP [9
Of studies examining COX
polymorphisms in association with breast cancer, few have examined interactions with NSAID use [11
]. Authors of only one previous study have included interaction analyses of aspirin and other NSAIDs separately [15
]. We observed a significant interaction of recent aspirin use with rs4648261. To our knowledge, interaction with this SNP has not been previously reported.
Findings for rs2745559 and rs2206593 were suggestive of interaction with both recent and adult lifetime aspirin use, although results were not statistically significant. Gallicchio et al. [11
] observed significant interaction between NSAID use and rs2745557 (a SNP in LD with rs2745559) in relation to breast cancer risk. Women carrying the homozygous variant of rs2745557 who were non-users of NSAIDs were at significantly increased risk of breast cancer compared to NSAID users carrying either the common genotype or heterozygotes [11
In sum, our findings of increased risk of breast cancer among carriers of the rs2745559 variant, and inverse associations with several other COX-2 SNPs, are largely consistent with published literature, including the recent findings of the BPC3. Although few have reported on interaction of COX-2 variation with individual NSAIDs, our findings of generally small interactions with aspirin use are consistent with those of others, although different assessments and considerations in statistical analyses limits comparability of studies. While others have looked at all NSAIDs together, we were able to look at aspirin separate from ibuprofen. We did not see any interaction with ibuprofen. It may be that differences between mechanisms of action for aspirin and ibuprofen explain differences observed for interaction models.
Several mechanisms by which polymorphisms may alter COX
function have been proposed, although little is known about their relation to phenotype. SNPs in the gene body may alter the COX-2 enzyme conformation, and therefore alter enzymatic function or the affinity of NSA-IDs to bind to the enzyme [32
]. In murine models, polymorphisms in the 5′ and 3′ untranslated regions (UTR) are thought to alter gene promoter activity or alter mRNA stability, respectively [27
]. The suggestion of a reduction in risk that we observed associated with rs2143416 and rs12042763, both in the 5′-UTR, may correspond to decreased COX
expression. The increase in risk we observed with the variant of rs2745559 (5′-UTR) may be due to altered gene expression or due to some other mechanism—including one involving the SNP for which it tags, rs2745557, located on intron 1. Currently, the functional impact of neither SNP is known. Further, chance cannot be ruled out as an explanation for our findings in that the observation was not statistically significant after adjustment for multiple comparisons. It could be that the polymorphic variants of rs5275 and rs2206593 both in the 3′-UTR may affect the transcript’s stability, therefore altering inflammation levels. The functional impact of rs4648261, located on intron 2, for which we observed significant interaction with aspirin use, is also not known. It may be that rs4648261 is in LD with a SNP in a translated region of the gene, or that intron 2 includes certain regulatory regions, or that the finding was a result of chance.
This study has several limitations. As very little is known about the function of these COX-2 variants, at this time we can only hypothesize regarding their biologic role. Since we had no a priori hypotheses regarding function, for the analysis of interaction, we determined the referent group based on the findings from the analysis of main genetic effects. That is, the referent was determined as the group who were non-users and with apparently higher OR for the main effects. With more information regarding function, we might use a different group as referent. Further, our understanding of other factors influencing gene expression is limited. It may be that these genetic variations act in concert with other factors such as epigenetic changes, and that to understand this process, we need to know more about those dimensions as well.
One concern in a study of this kind is power. For most SNPs, we had 80% power to detect ORs of ≤0.79 or ≥1.25 using a dominant model, and interaction ORs of ≥0.62. We cannot rule out associations among very rare SNPs that we did not have power to detect in our study. Inherent to genetic analyses such as these, we do not know if the SNPs studied are the true risk alleles, or rather linked to other polymorphisms of biologic significance. One SNP, rs5277, was out of HWE in our study population and we were therefore unable to study that haplotype block and cancer risk. While population stratification is a concern in genetic association studies, it is not likely to have had a large impact on our findings as our analysis was restricted to Caucasians [35
It is possible that participants’ recollection of NSAID use was dependent upon case–control status. Given the design of our study, we believe it is unlikely that the interviewers or participants were aware of NSAID use as a study hypothesis and probing of participants or over-/under-reporting of exposures is not likely to have been differential. Further, our results were similar to those of prospective studies where recall bias is not an issue [2
]. In addition to possible recall bias in the report of NSAID use, there was likely non-differential error in the recall of participants’ lifetime aspirin use and recent NSAID use. Non-differential measurement error would result in widened confidence limits and attenuated point estimates [36
]. Although it is possible that participants could self-select based upon their use of NSAIDs, selection bias would not explain the observed gene–environment interaction [37
This study has several strengths. It is among the largest to investigate associations of COX
variation with breast cancer risk. Additionally, it is among the first to characterize this variation across such a large area of the gene. Previous investigations of COX
and cancer risk have captured variation across the gene body alone [9
] or only a small (≤2 kb) area surrounding the gene [31
]. As variation in the flanking region of COX
is hypothesized to contribute to gene function [27
], investigations including these regions are important. Only the BPC3 has reported on a larger area surrounding the gene [18
]. Another strength is that we were able to investigate gene–environment interaction, taking into account possible differences between aspirin and ibuprofen. In addition, this study is the first to explore gene–environment interaction examining more than recent use; we had reports of adult lifetime aspirin use.
In this study, we found a modest increase in risk with the variant of rs2745559. There was some indication that aspirin use among carriers of the rs4648261 variant allele may have additional chemopreventive benefit with regard to breast cancer. Epidemiologic studies with large sample sizes and careful examination of exposure to NSAIDs are needed to further assess associations of variation in COX-2 and other genes that impact inflammation with breast cancer risk and to investigate possible interaction with aspirin and NSAID use. Further study of the function of COX-2 polymorphisms is warranted to better understand what may be an important mechanism for carcinogenesis in general and for breast cancer in particular. Better understanding of the role of inflammation in breast carcinogenesis has potential to inform prevention strategies.