1. Introduction
Colorectal cancer (CRC) is the third most common malignant tumor and the fourth leading cause of cancer death worldwide with a lifetime risk in Western European and North American populations of around 5% [
1]. Multiple risk factors, both genetic and environmental, are involved in the etiology and prognosis of CRC [
2]. Identification and characterization of the risk factors, their potential interactions, and the underlying biological mechanisms are requested as a basis for improving preventative strategies that may include identifying individuals who would most benefit from preventive strategies.
Epidemiological studies suggest that the high intake of red and particularly processed meat may increase the CRC risk [
3], whereas long-term use of non-steroid anti-inflammatory drugs (NSAIDs) including aspirin (acetylic acid) may reduce the risk of CRC [
4,
5]. Investigations on the potential carcinogenic mechanisms of red and processed meat have suggested that meat may confer carcinogenesis by being a source of cooking mutations (heterocyclic amine,
N-nitroso compounds) formed during preparation [
6], organic sulphur-containing proteins leading to a high content of H
2S in the intestinal lumen, a highly potent regulator of intestinal cell function including inflammation and cell death signalling [
7] and/or microbial factors arising during storage [
8]. Similarly, the underlying cancer protective mechanisms of NSAID have been investigated and both COX-2 dependent and COX-2 independent mechanisms have been suggested [
9,
10].
Still, however, the mechanisms are incompletely understood. First of all, epidemiological studies are not suitable to evaluate CRC causality because of collinearity between the studied factors (intake of red and processed meat and NSAID) and other potential CRC risk factors (such as e.g., Western-style diet and high body mass index) that limit the ability to analytically isolate the independent effects of the studied factors [
11]. Next, although animal studies may suggest important underlying biological mechanisms [
12], results from animal studies may not apply to humans due to differences in the biology such as the metabolism of meat between animals and humans and because doses used in animals may not be transferable to human conditions [
6]. Gene-environment (GxE) interaction analyses may overcome the methodological issues mentioned above. Indeed, the identification of an interaction between a genetic variant (functional or in linkage with a functional variant) in a gene that is chosen based on its biological function and an environmental factor suggests that both factors are involved in the same process. Using GxE interaction analysis, we have investigated potential mechanisms by which red and processed meat and NSAID may affect CRC carcinogenesis [
13,
14,
15,
16,
17,
18,
19,
20,
21] (reviewed in [
22,
23,
24]). Red and processed meat is a rich source of n-6 polyunsaturated fat that is converted into arachidonic acid after ingestion and further metabolized into several bioactive lipids that play critical roles in a variety of biologic processes involved in chronic inflammation and colorectal cancer. Conversely, NSAIDs including aspirin may reduce inflammation and CRC risk via similar and other pathways in relation to CRC [
22,
23,
25].
Thus, the aim of the present study was to investigate the association of polymorphisms in genes involved in fatty acid metabolism and NSAID pathway with CRC, and, furthermore, interactions between these polymorphisms and NSAID use and dietary factors focusing on the intake of red and processed meat in relation to CRC. The study cohort was the Danish “Diet, Cancer and Health” with prospectively collected lifestyle information encompassing 57,053 participants, whereof 1038 cases that developed CRC were compared to a sub-cohort of 1857 members using a nested case-cohort design. In addition to replicating earlier findings, this study found interactions between genetic variants in fatty acid metabolism and NSAID pathway and intake of red and processed meat suggesting that meat intake and NSAID use affect the same carcinogenic mechanisms.
3. Discussion
This large prospective study investigated potential associations between polymorphisms in the fatty acid metabolic and NSAID pathways, and risk of CRC and, furthermore, the potential interaction between these polymorphisms and NSAID and diet (intake of red and processed meat, fiber, fruit and vegetables, and alcohol) in relation to CRC. The polymorphisms were selected from recent reviews based on their potential role in the fatty acid metabolic and NSAID pathways (
Table 5) [
22,
23,
24,
26]. We found that
CCAT2 rs6983267 GG genotype was associated with lowered risk of CRC per se and we found an interaction between the polymorphism and meat in relation to CRC. Furthermore, interactions between
TP53 rs1042522 and use of NSAID, alcohol intake, and, in the tertile analysis, intake of red and processed meat was found. Next, we found interactions between
LPCAT1 rs7737692 and
SLC25A20 rs7623023 and intake of red and processed meat in the tertile analysis in relation to CRC. No other consistent associations or interactions were found.
First, the association of
CCAT2 rs6983267 with CRC confirmed earlier results from several independent populations [
25,
31] supporting the importance of the 8q24.21 gene locus for CRC carcinogenesis. The
CCAT2 rs6983267 polymorphism is located in a non-protein coding region near the
MYC gene. The T-allele of
CCAT2 rs6983267 has been shown to impair binding of WNT/CTNNB1 pathway-related transcription factor 7 like-2 to DNA, thereby reducing
MYC expression, which, in turn, induces resistance to intestinal tumorigenesis [
25]. The polymorphism has also previously been found to interact with aspirin. Nan et al. found that variant T-allele carriers had 39–48% lower risk of CRC while using aspirin [
25]. T-allele carriers of
CCAT2 rs6983267 constitute 27% of the sub-cohort members in the present study. As we did not find an interaction between
CCAT2 rs6983267 and NSAID use in the present study, the result may potentially suggest a specific effect of aspirin that may not be shared with non-aspirin NSAIDs in general. Unfortunately, the present study did not have the power to investigate aspirin use only.
Next, we found an interaction between
TP53 rs1042522 and NSAID. In our study, GG homozygotes lowered their risk of CRC by use of NSAID, whereas variant C-allele carriers increased their risk of CRC by NSAID use (
p = 0.04). This is a replication of an earlier finding [
32]. Tan et al. observed that GG homozygotes benefitted more from the use of NSAID than variant C-allele carriers. They found a substantial protective effect of NSAID use for homozygous carriage of the 72Arg allele compared to the 72Pro allele (odds ratio 0.44; 95% CI: 0.30–0.65) [
32].
In the present study, four polymorphisms (
CCAT2 rs6983267,
TP53 rs1042522,
LPCAT1 rs7737692, and
SLC25A20 rs7623023) were found to interact with meat intake (
Table 4 and
Table S1). Two of the polymorphisms (
SLC25A20 rs7623023 and
LPCAT1 rs7737692) are involved in the metabolisms of fatty acids (
Table 5); however, the functionality of the two common polymorphisms is unknown. The protein coded by
LPCAT1 is involved in the remodelling of phospholipids and has been associated with risk of sudden cardiac arrest [
26], whereas the protein coded by
SLC25A20 is involved in the transport of fatty acids across the mitochondrial membrane. Our results may suggest that the fat from red and processed meat (that is metabolized to fatty acids) may contribute to the carcinogenic mechanism of red and processed meat in relation to CRC.
The two other polymorphisms (
CCAT2 rs6983267 and
TP53 rs1042522) have been found to interact with aspirin/NSAID in relation to CRC in the present or other studies [
25,
32].
TP53 rs1042522 is a missense polymorphism in the
TP53 gene where Arginine is changed to Proline, which results in increased apoptosis potential due to increased p53 levels [
27,
28]. This polymorphism has been found to be associated with many different cancer types including breast cancer, lung cancer, endometrial cancer, non-Hodgkin lymphoma, esophageal squamous cell carcinoma, bladder cancer, ovarian cancer, neuroblastoma and hepatocellular carcinoma supporting its functionality. Several epidemiological studies, including randomized controlled clinical trials, have demonstrated that NSAID use decreases the incidence of adenomatous polyps and CRC [
5]. The mechanism is thought to be caused by cell-cycle regulation and/or induction of apoptosis via mechanisms dependent and independent of cyclooxygenase [
5,
33]. The use of NSAID may enhance the apoptosis potential already present in the GG genotype of
TP53 rs1042522 resulting in decreased risk of CRC compared to variant C-carriers. A diet high in meat was associated with increased risk of CRC among variant C-allele carriers compared to those with a diet low in meat intake. We have previously shown that intake of meat interacts with polymorphisms in inflammatory genes in relation to CRC risk [
17,
18,
34], suggesting that a diet high in meat may cause an inflammatory milieu that increases the carcinogenic potential in persons with an impaired
TP53 gene. This hypothesis could also apply for the
CCAT2 rs6983267 polymorphism since persons homozygous for the G-allele have a higher expression of
MYC [
29] and thereby an increased carcinogenic potential, which could be further triggered by a diet high in meat. The finding that alcohol intake interacted with
TP53 rs1042522 resulting in an increased risk of CRC for variant C-carriers may be caused by a similar mechanism as meat since alcohol is known to be associated with a systemic inflammatory state [
35] and thus the protective effect of the G-allele is abolished.
Advantages and limitations with the study design have been described in previous studies [
15,
16,
17,
18,
19,
20,
21]. The main advantage of this study is the prospective study design with the collection of dietary and lifestyle factors before diagnosis that eliminates the risk of recall bias. Another main advantage is the diverse and high intake of meat in the present cohort enabling identification of gene–meat interactions. The prospective “Diet, Cancer and Health” cohort has proven to be suitable to detect meat–gene interactions [
17,
18,
34]. Changes in dietary and lifestyle habits during follow-up is possible, but, if present, will result in lower power to detect real differences between cases and the comparison group. The “Diet, Cancer and Health” cohort is homogenous reducing population specific genetics and dietary patterns seen in larger multicentre studies. The disadvantage of the prospective study is the limited power to study gene–environment interactions. None of the results withstood Bonferroni correction. Thus, all new findings should be replicated in independent prospective cohorts with well-characterized lifestyle information.