In this nested case-control study the association between 8q24 SNPs and prostate cancer is modified by pesticide use. In particular, the association between rs4242382 and prostate cancer is increased across strata of fonofos, terbufos, and permethrin exposure. Furthermore, the joint effect of fonofos exposure and carriage of three or four risk alleles in rs7837328 and rs4242382 resulted in a 3-fold increased risk of prostate cancer risk compared to subjects who had zero risk alleles and had never used fonofos. The association between several region 3 SNPs and prostate cancer also appeared to be modified by several pesticides including coumaphos and permethrin, and increased risks associated with rs12547643 (telomeric to regions 3 and 1) across strata of other chemicals are also evident.
The observed associations for prostate cancer among 8q24 variants in our study are consistent with GWAS reports. Similar to several reports, we observed a stronger association for rs4242382 compared to rs1447295 in region 1 and risk of prostate cancer. (5
) The independent risk marker rs6983267 in region 3 (9
) was also associated with prostate cancer, although other correlated variants in regions 3 showed stronger signals in our study. Additionally, the reported prostate cancer susceptibility loci rs445114 and rs620861 were also associated with prostate cancer risk, however, these were not among the strongest signals. Another locus, rs12547643, telomeric to regions 3 and 1, was found to be associated with prostate cancer in our study. This marker lies in a separate block of linkage disequilibrium from the other regions and is close to a marker, rs9642880, which has been associated with susceptibility to bladder cancer. (26
) Although the association between rs9642880 and prostate cancer was not significant in our study (p-trend=0.153), the pairwise correlation between rs9642880 and rs12547643 is not negligible (r2
=0.20). This variant however, has not been shown to be associated with prostate cancer in GWAS (p=0.77) (11
), thus the observed association in our study may be due to chance. GWAS SNPs from region 2 were not genotyped in this study.
In the AHS we have observed the association between prostate cancer and pesticides to be modified by family history of prostate cancer (19
), which could be a marker of genetic susceptibility. Some of the chemicals that are shown here to modify the association between 8q24 variants and prostate cancer have also been associated with prostate cancer among those with a family history in pesticide-specific analyses within the AHS (19
), and include fonofos, phorate, coumaphos, and permethrin. Thus, we also explored whether family history of prostate cancer was related to 8q24 variants. Two studies have reported a potential association between region 1 SNPs, rs4242382 and rs1447295, and family history of prostate cancer, though both studies are noted to be underpowered due to the limited number of cases and controls with a family history of prostate cancer. (32
) Larger GWAS reports have reported no significant interaction between rs1447295 or rs6983267 and family history prostate cancer (9
) although not all GWAS loci have been evaluated. We did not observe an association between region 1 variants and family history of cancer. We did observe a greater proportion of subjects with risk alleles in region 3 variants among those with a family history (data not shown), however, the proportions among cases and controls were similar and adjustment for family history in all interaction models did not change the risk estimates. When we restricted analyses to those with no family history of prostate cancer all results persisted. Thus, the previously observed effect modification of family history on the association between pesticides and prostate cancer does not appear to be explained by variants on chromosome 8q24. It is possible that other genes, multiple genes, or non-genetic factors that track in families might account for this previously observed association.
Several pesticides of similar chemical structure were observed to modify the association between 8q24 SNPs and prostate cancer. The organophosphate insecticides, coumaphos, fonofos, phorate, and terbufos consistently modified the association between regions 3 and 1 SNPs and prostate cancer. The strongest association is for fonofos where the risk of prostate cancer was over three times as likely per rs4242382 risk allele among high users. Among ever users of fonofos, subjects with 3 or 4 risk alleles in rs7837328 and rs4242382 had 3.14 times the risk of prostate cancer compared with subjects who had zero risk alleles and had never used fonofos. Previous analyses revealed the strongest effect modification for any particular pesticide and family history was for fonofos. (21
) Thus, the risk of prostate cancer among fonofos users may be especially important among genetically susceptible subgroups. Ten OP chemicals were examined and results for coumaphos, phorate, and terbufos were similar to those observed for fonofos although not statistically significant. Fonofos, coumaphos, phorate, and terbufos are all classified by the U.S. Environmental Protection Agency as Group E for carcinogenicity (evidence of non-carcinogenicity for humans) based on the absence of mutagenic, genotoxic, and carcinogenic observations in limited experimental and animal tests. (35
) In 1998, the registrant of fonofos voluntarily cancelled its registration (36
) while the other OPs are still in widespread agricultural use.
Although interactions were not statistically significant after correction for multiple testing, several other pesticides appeared to modify the association between 8q24 SNPs and prostate cancer. The increasing pattern of risk across permethrin exposure strata is similar to that observed for the four OPs, possibly due to the fact that it is also an insecticide, although not all insecticides examined modified risk. Several other compounds with chlorine substituents including captan, chlordane, dieldrin, and metolachlor appeared to modify risk with rs12547643 (telomeric to regions 3 and 1). We have previously reported that AHS applicators over the age of 50 who used chlorinated pesticides had an increased risk of prostate cancer. (19
) It is unclear whether the chlorinated components of these chemicals are relevant to risk or whether this SNP is truly associated with prostate cancer. Thus, it is possible that these trends across strata may be due to chance.
While some similar chemicals were observed to interact with the 8q24 region suggesting a common biological mechanism of action, the precise mechanism is unclear. Little is known overall about whether or how pesticides may be associated with cancer; however, substantial literature documents the principal involvement of phase I and phase II enzymes in the metabolism of specific xenobiotic substrates, including pesticides. (37
) Most organophosphate insecticides (including coumaphos, terbufos, fonofos, and phorate) must be activated to their oxon (potent cholinesterase inhibitors) to be excreted. The oxon form of these compounds has been associated with a number of biological endpoints including neurotoxicity, the generation of reactive oxygen species, and DNA damage. (39
) Currently however, there is no evidence to suggest that variation in the 8q24 region impacts xenobiotic metabolism. Other evidence indicates that at least one of the 8q24 variants resides in an androgen receptor transcriptional enhancer site, suggesting a potential hormone-related mechanism. (40
Recent studies have explored whether 8q24 variants might be related to the nearest coding region, the MYC
gene and its expression. Two studies show that rs6983267 is located in a transcriptional enhancer and affects a binding site for TCF4, a transcription factor which interacts with β-catenin to activate transcription of Wnt target genes. (15
) It was also demonstrated that a DNA restriction fragment containing rs6983267 physically interacts with the MYC
promoter in colorectal cancer cell lines. (16
) Imbalances in Wnt-mediated regulation of MYC
are associated with altered cellular adhesion, proliferation, and differentiation. (12
) In addition, experimental animal studies have also shown that Wnt signaling can be altered upon exposure to OPs (42
) and chlorinated pesticides (43
) and that pesticides can influence MYC expression as well. (45
) Thus, this may be a potential mechanism by which genetic variation in 8q24 modifies the risk associated with pesticide use and suggests a role for enhanced alterations in important global cancer signaling pathways.
Several strengths and limitations of our study should be recognized. High quality genotype and pesticide information is available in the AHS. For many gene-exposure studies, the key limitation is the quality of the exposure information. The information on pesticide use among AHS participants is superior to any other epidemiologic studies of cancer. This is because farmers are aware of the type and duration of pesticides they use as these are often restricted use chemicals for which a license is required; self-reported pesticide use information has been found to be reliable in this cohort. (48
) Furthermore, the ability of the AHS to look at individual pesticides rather than groups (herbicides or insecticides or chemical classes) is critical because observed cancer risks appear to be chemical-specific. Under this premise, we chose to treat each pesticide as independent in the multiple comparison correction which results in a less conservative correction. Thus, the highlighted results for fonofos interactions, like all the interactions presented in this study, would need to be confirmed to see if they are truly important for prostate cancer. In addition, the numbers within some pesticide use strata were small, however, to our knowledge there are no other studies with more power to examine this interaction. Also, we were not able to examine disease aggressiveness due to small numbers. Finally, subjects in this study were all Caucasian, which limits the generalizability of the results to other racial/ethnic groups.
In conclusion, we observed an interaction between variants on chromosome 8q24, pesticide use, and risk of prostate cancer. Insecticides, particularly several organophosphates, appear to be the strongest modifiers of risk. This is the first report of effect modification between an 8q24 region variant and environmental exposure on prostate cancer risk. These results need to be subjected to replication due to the high probability of false positive results from the multiple tests of interaction. Opportunities for replication, however, are currently limited because of the lack of comparable agricultural populations with sufficient sample size, detailed information on pesticide use, as well as DNA for genetic analyses. We continue, however, to look for possible studies in which to replicate these findings. Further research should continue to explore the possible mechanism or mechanisms for pesticide induced prostate carcinogenesis because if the current interactions are indeed true, this could provide critical new information about a previously unsuspected biological pathway through which prostate carcinogenesis occurs. Similarly, mechanistic studies that help identify the role of the 8q24 region on prostate cancer risk may also offer critical new clues to help explain prostate carcinogenesis.