Our findings offer the first comprehensive analysis of the interaction between HCA intake, genetic polymorphisms in xenobiotic metabolizing genes that are known to metabolize these compounds and prostate cancer risk. We observed that the effect of HCA intake on the risk of prostate cancer differs by GSTM3 and GSTP1 genotypes in particular; interactions with the EPS8L3 Pro356Ser polymorphism (rs11102001) just downstream of the GSTM3 locus were statistically significant at the FDR = 0.20 level and the GSTP1 Ile105Val polymorphism (rs1695) also appeared to modify risk.
We observed suggestive HCA interactions with 3 SNPs located in the GSTM3
region (rs2274536, rs1887546, rs11102001). All three of these SNPs are located within the 10kb downstream margin of GSTM3
and are located in another gene called EPS8L3
, epidermal growth factor receptor pathway substrate 8-like 3. This gene, and other member of the EPS8
), encodes proteins that are responsible for Ras to Rac signaling leading to actin remodeling or cytoskeletal integrity (30
). The Ras signaling pathway regulates normal cell proliferation. Ras and Ras-related proteins are often deregulated in cancers, leading to increased invasion, metastasis, and decreased apoptosis (33
). Ras activation is a component of the signaling pathways for virtually all the receptors shown to be upregulated in advanced prostate cancer (34
). The polymorphic site at codon 356 in exon 12 (rs11102001), where a guanine-to-adenosine (G-A) transition occurs, causes a proline to serine substitution and had a minor allele frequency of 7% among controls in our population, which is consistent with the minor allele frequency observed in HapMap for Caucasians (7%). While no literature describes any loss of activity associated with this polymorphism or that this amino acid substitution is likely to be damaging (PolyPhen‡
prediction score = 0.286), carriers of the variant allele (A) with high MeIQx and DiMeIQx intake were at higher risk for prostate cancer compared to those with low intake. A recent study showed that PhIP stimulates cellular signaling pathways and resulted in increased growth and cell migration in human mammary epithelial cell lines (35
). Thus, increased HCA exposure could similarly act as a promoter of malignant transformation by increasing mitogenic signaling.
Alternatively, the mechanism of action responsible for the observed effect might be linked to xenobiotic metabolism pathways. GSTM3
is expressed in prostate tissues (36
) and acts to detoxify active HCA metabolites by conjugation with glutathione (27
). Altered expression of the enzyme could lead to differential clearance of activated HCA metabolites resulting in an accumulation of DNA damaging species, which could increase the risk for carcinogenesis at this site. The three EPS8L3
SNPs are located just downstream of GSTM3
and show strong linkage disequilibrium with variants in the GSTM3
locus; therefore, it is possible that these SNPs are surrogate markers of other variants in the GSTM3
locus that were not genotyped. Alternatively, these downstream variants could alter GSTM3
expression. After adjustment using the FDR method, the MeIQx-rs11102001 and DiMeIQxrs11102001 interactions appear to be the most interesting findings, suggesting a real modification of prostate cancer risk. Thus, further examination of variants in this region is warranted.
HCA intake was estimated from a meat-cooking FFQ module, which included questions about meat type, cooking method, and cooking time, or ‘doneness’ level, used in conjunction with a HCA database. HCA formation in meat generally increases with temperature and doneness level. PhIP is the most abundant HCA, then MeIQx and DiMeIQx is the least abundant; IQ (2-amino-3methylimidazo[4,5-f]quinoline) and MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline are not typically detected in meat samples (38
). The questionnaire and procedure for estimating HCA intake has been validated and found to be an acceptable method for HCA exposure assessment (39
We observed an effect for DiMeIQx and MeIQx in the presence of GSTM3
variants. Although DiMeIQx is consumed at a low level with respect to other HCAs in the diet, DiMeIQx and MeIQx are observed to be more potent mutagens than PhIP (40
) thus their impact on carcinogenesis may be greater. There is little information in the literature with respect to the activity of GST enzymes in the detoxification of DiMeIQx and MeIQx. As the formation of PhIP is the highest, the pathway for detoxification of this compound is better described with respect to several Phase II enzymes (27
). Information on DiMeIQx is lacking with respect to detoxification pathways and carcinogenicity data, although it is known to be mutagenic in bacterial assays. The continued evaluation of DiMeIQx and MeIQx is needed as more studies of HCAs and cancer risk implicate these more potent HCAs.
We also observed lesser effects for HCA interactions with one promoter polymorphism (rs6591256, position -1415) and one nonsynonymous polymorphism (rs1695, isoleucine to valine substitution) in GSTP1. GSTP1
is the major GST identified in benign prostate hyperplasia tissue samples relative to other GST enzymes (37
). In human prostate tissue, however, expression of this enzyme is silenced via hypermethylation of the promoter region (42
), occurring in approximately 90% of prostate adenocarcinomas (42
) suggesting that alteration of GSTP1
activity is related to prostate carcinogenesis. The polymorphic site at codon 105 in exon 5, where an adenosine-to-guanine (A-G) transition causes an isoleucine to valine substitution (rs1695), has also been extensively described (44
). The valine substitution results in lower enzyme activity (45
) for certain substrates and thus may impair detoxification of carcinogens. While these interactions were not statistically significant, consideration of both genetic and environmental exposures may offer additional insight into our understanding given the known biologic impact of altered GSTP1
expression in prostate carcinogenesis.
Although we observed some modifying effects in SNPs in the UGT1A
locus and in CYP1B1,
the magnitude of these findings was not as large as those for GSTM3
. We also did not observe a strong modifying effect for SNPs in CYP1A1, CYP1A2, GSTA1, GSTM1, NAT1, NAT2, SULT1A1
, and SULT1A2
. The lack of findings in these genes does not mean that they do not play a role in altering risk via differential HCA metabolism. Variants in CYP1A2
and in the NATs
have been well described in HCA metabolism and often implicated to potentially alter cancer risk (47
is the principal hepatic CYP involved in the bioactivation of HCAs to their N-hydroxy metabolites and NATs
are responsible for further bioactivation of N-hydroxy-HCA metabolites to genotoxic N-acetoxy-HCA esters (29
). It is possible that we did not find an association for these genes because the bioactivation process is less important than the detoxification processes performed by the GSTs or that expression of these enzymes in the target tissue, the prostate, is lower. However, it is also possible that important SNPs in these genes and others were not genotyped or captured by our tagSNPs or that the power to detect these associations was low.
Strengths of our study included a relatively large sample, a detailed assessment of dietary HCA intake, and a comprehensive characterization of the genes involved in HCA metabolism. Given that this study was nested within the screening arm of the PLCO trial, selection and surveillance bias is minimal because both cases and controls had equal opportunity to be detected with prostate cancer. While this is the largest study to evaluate the interaction between dietary HCA intake and genetic variants in XME genes and risk for prostate cancer, the presence of low frequency variants limits the power to detect significant interactions. Thus, we have attempted to identify interesting associations that need to be followed up in future studies. Despite the potential limitation in power, it was important to characterize the top HCA intake category using the highest quintile of exposure based on previous positive associations with PhIP and prostate cancer only in the top quintile of intake. This study is comprised of only non-Hispanic Caucasian men, which limits the generalizability of our findings to other race/ethnic groups with different genetic compositions; however, as a result of the racial homogeneity, population stratification is unlikely to be a significant source of bias in this study (49
Data from genome wide association studies have yielded novel and interesting insights into genetic risks for prostate cancer. These types of studies, however, do not take into consideration the complex interaction between genetic variants and environmental exposures. In conclusion, variants in two genes known to detoxify HCAs, GSTM3/EPS8L3 and GSTP1, modify the association between dietary HCA intake and prostate cancer. Despite the fact that genome wide scans have not identified these genes as important prostate cancer risk factors in main effect studies, additional studies with more power should continue to evaluate these genes in relation to environmental interactions and prostate cancer.