Analysis of potentially functional polymorphisms in candidate genes has emerged as a powerful approach in deciphering the complex relationship between genotype and phenotype. In this context, association analyses can be used to explore the role of genetic polymorphisms in susceptibility to various cancers and response to specific chemopreventive agents. In our study, analysis of four haplotype-tagging SNPs in the regulatory regions scattered over the entire Cox-2 gene revealed important aspects of its allelic structure with consequences for adenoma risk and interactions with NSAIDs.
Cox-2 is an inducible enzyme and its expression and stability appear to be subjected to very complex mechanisms regulated by various elements in both the 5′UTR and 3′UTR of the transcript as well as intronic sequences (Dixon et al, 2000
; Kirtikara et al, 2000
; Finkbeiner, 2001
; Tanabe and Tohnai, 2002
; Konheim and Wolford, 2003
). Several promoter elements, such as CRE as well as those specific for binding to a variety of nuclear regulatory factors including Nfκ
B, NF-IL6, and myb, may play an important regulatory role in the transcription of the Cox-2
gene in a tissue-specific manner (Potter et al, 2000
; Mestre et al, 2001
; Tang et al, 2001
). Polymorphisms may either eliminate or create binding sites for various factors potentially altering the expression of Cox-2 and thereby modulating the risk for various cancers. Recently, four SNPs in the promoter region of Cox-2
(−297, −899, −1265, −1285), different from the ones used in our study, have been described to modulate the risk for prostate cancer in both African Americans and Caucasian individuals (Panguluri et al, 2004
We examined two promoter polymorphisms, −663 and −798, the Cox-2 gene which are not located in binding sites of any known transcription factors. The homozygous variant at −663 was extremely rare in our study cohort, but the heterozygous genotype was clearly protective for adenoma risk in males (). Our data also show that the variant allele at intron 5-5229 reduced the risk for adenoma development in males and, when analysed in the context of haplotype, neutralised the risk effect of the 3′UTR-8494 variant allele. Therefore, either the intron 5-5229 polymorphism itself or another polymorphism that is in linkage disequilibrium with intron 5-5229 in the Cox-2 gene may be relevant for its transcriptional regulation and/or stability.
Besides the promoter and intronic sequences, elements in the 3′UTR play an important role in polyadenylation, nuclear export, degradation, stabilisation, and translation of the transcripts (Kuersten and Goodwin, 2003
). Binding of translational regulatory factors to ARE (AU-rich element) in 3′UTR has been shown to alter Cox-2 expression (Dixon, 2004
). In vitro
experiments have shown that overexpression of trans-activating cellular factors, which bind to Cox-2
ARE, stabilise mRNA resulting in increased expression of Cox-2 in colon cancer cells (Dixon et al, 2000
). Conceivably, polymorphisms in the 3′UTR of Cox-2
may modify the binding affinity of regulatory factors and influence its stability and expression. The 3′UTR-8494 polymorphism analysed here is located in the AU-rich region that mediates transcript degradation. Our finding that the 8494 variant allele increases the risk for colorectal adenomas in males by 31% suggests that the polymorphism has a transcript-stabilising function. Interestingly, the same 3′UTR-8494 polymorphism was recently reported to carry an enhanced risk for lung cancer (Campa et al, 2004
), suggesting its significance in the regulation of Cox-2
transcripts and the subsequent impact on multiple cancers. However, the same polymorphism did not show an effect on colorectal cancer in another recent report, although two downstream SNPs were associated with an increased risk (Cox et al, 2004
). This lack of association of the 3′UTR-8494 polymorphism with colorectal cancer risk (Cox et al, 2004
) could be due to relatively small number of subjects (292 cases/274 controls) and/or analysing males and females together. In any event, association of the 3′UTR-8494 variant and/or nearby polymorphisms in colorectal and lung carcinogenesis warrant a closer look at this region for its role in post-transcriptional regulation of the Cox-2
gene with implications for colorectal and lung carcinogenesis.
In our study, sequence variations in the Cox-2
gene, with a potential to affect its expression and/or stability, appear to modulate the risk for adenoma development only in males. Previous studies have documented gender differences in the incidence, location, and pathogenesis of colonic adenomatous polyps and tumours (DeCosse et al, 1993
; McCashland et al, 2001
; Weissfeld et al, 2005
). The reduced colorectal cancer risk in women has been attributed to physiological, environmental, and behavioural factors (DeCosse et al, 1993
). Especially, oestrogen has been suggested to have a protective role in colon carcinogenesis by affecting metabolism of bile acids and serum levels of insulin-like growth hormone levels (Everson et al, 1991
; Campagnoli et al, 1993
). Transcriptional silencing of the oestrogen receptor gene has been reported in colorectal tumours (Issa et al, 1994
) and a meta-analysis of several epidemiological studies of postmenopausal women reported a significant reduction in the risk of colorectal cancers with hormone therapy (Grodstein et al, 1999
). The apparent lack of any role of Cox-2
polymorphisms in the development of adenomas in females noted in our study could simply be due to relatively small numbers analysed and certainly needs further validation. A comprehensive analysis of various Cox-2 haplotypes in a much larger case–control study comprising of females would help clarify this issue.
Another important aspect of our analysis is that it highlights the significance of the overall allelic architecture of Cox-2 on disease risk rather than its effect based on individual SNPs in isolation. Among the four polymorphisms analysed, the combination and/or interplay between two SNPs (minor allele frequencies of 0.35 and 0.43 for intron 5-5229 and 3′UTR-8494, respectively) were important in determining the risk of developing colorectal adenomas. The allelic influence of these two individual polymorphisms conferring either risk of or protection from adenoma development was neutralised in individuals carrying both variants ().
Cyclooxygenase 2 is a primary target for NSAIDs, which have been shown to reduce the risk of colon cancers and colorectal adenomas. Aspirin use was associated with a decrease in the recurrence of colorectal adenomas in randomised clinical trials (Baron et al, 2003
; Benamouzig et al, 2003
; Sandler et al, 2003
). Sequence variations in the Cox-2
gene could conceivably modify the chemopreventive effect of aspirin (Ulrich et al, 2005
) and possibly other NSAIDs. In our analysis, the Cox-2
haplotype with wild-type alleles of intron 5-5229 and 3′UTR-8494 (also of −663 and −798, data not shown) was pharmacologically beneficial in males (). The GC haplotype carrying the protective and risk alleles was also suggestive of a beneficial effect in male aspirin users. On the contrary, not only that females do not appear to drive any benefit from aspirin use, female carriers of the GC haplotype were at a significantly increased risk for adenoma development. Of cautionary note here is, the increased risk, although significant, was based on small number of female aspirin users with the GC haplotype.
The gender difference has also recently been reported in the cardiovascular response to aspirin. The risk of myocardial infarction or death from cardiovascular causes in women was not altered by aspirin but was significantly reduced in men (Steering Committee of the Physicians' Health Study Research Group, 1989
; Levin, 2005
; Ridker et al, 2005
). Our preliminary observation of the harmful effect of aspirin on colorectal adenoma in females, if confirmed in a larger study, would be helpful in pharmacological stratification of patients according to the Cox-2
Cyclooxygenase 2 is a crucial enzyme in a key signalling pathway and has been the target of prevention/intervention strategies in many clinical trials. Selective inhibition of Cox-2 results in variable responses in individual patients. An exhaustive approach using the haplotype-defining SNPs in the gene as well as information on the functional significance of polymorphisms with the risk-modulating ability would have significant implications not only for risk identification but also for pharmacological management of the disease.