The micronutrient Selenium (Se) is essential for human health and sub-optimal intake has been suggested to increase risk of various multifactorial diseases [1], [2]. Increased dietary intake of Se has been proposed to lower cancer mortality [3] and in particular Se has been reported to have a protective effect against prostate cancer [4], based partly on the results of a trial in the US that found an additional 200 µg Se/day to lower prostate cancer incidence in individuals who had relatively low Se status prior to supplementation [5]. However, a second supplementation trial (SELECT) failed to confirm this observation [6]. Although the different outcomes of these trials are likely to be due to a higher baseline Se status in the more recent SELECT study [7], they may also be affected by differences in the characteristics of the probands, such as pattern and prevalence of Se-related genetic variants in the study cohorts.

The biological functions of Se are carried out primarily by selenoproteins which contain Se in the form of the amino acid selenocysteine [8] and it is likely that the anti-carcinogenic properties of Se are brought about through these selenoproteins [9]. The selenoproteins have functions in cellular antioxidant protection (glutathione peroxidases, selenoproteins W and H), redox control (thioredoxin reductases), Se transport (selenoprotein P), and the endoplasmic reticulum unfolded protein response (selenoprotein S, 15 kDa selenoprotein, selenoprotein K) [10]. GPx3 and selenoprotein P (SePP) are secreted into the bloodstream and their plasma level, as well as serum Se, are commonly used as markers of Se status [11], [12]. A functional interaction between selenoproteins and prostate cancer has been reported, i.e. serum Se and selenoprotein P (SePP) concentrations are reduced in prostate cancer patients and this is correlated with disease severity [13]. This in turn could reduce selenoprotein expression and associated anti-oxidant defense resulting in increased oxidative damage leading to prostate cancer progression [14].

Selenocysteine incorporation into selenoproteins occurs during translation and requires proteins such as SECIS-binding protein 2 (SBP2) [7], [10]. Genetic variants in genes encoding the selenoproteins or components of the selenocysteine incorporation machinery would be expected to influence the biological pathways that are modulated by selenoproteins [15], [16]. Indeed, functional single nucleotide polymorphisms (SNPs) have been identified in a number of selenoprotein genes [13], [14] and disease association studies have linked variants in SEPP1, GPX1, GPX4, SEP15 or SELS to risk of various diseases [15], [16]. Three recent studies have studied the association of single selenoprotein gene variants with prostate cancer risk [13], [17], [18] and importantly they suggest that interactions between different SNPs in selenoprotein genes or antioxidant protein genes and Se status may influence susceptibility to prostate cancer or disease mortality. The common mechanism by which Se is incorporated into selenoproteins, the hierarchy of selenoprotein synthesis when Se supply is limited and the related functions of several selenoproteins (e.g. in redox control and unfolded protein response) all emphasise that selenoproteins are components of an integrated metabolic pathway. This close relationship between selenoproteins suggests that the influence of genetics on selenoprotein function and related disease risk is complex involving multiple interacting variants and both genetic and nutritional factors. However, such a comprehensive study of SNPs throughout the “Se pathway” in relation to prostate cancer has not been carried out.

As the selenoprotein family and selenoprotein biosynthesis pathway are well characterised, the aim of the present study was to investigate the association between SNPs throughout the genes encoding selenoproteins, factors essential for selenocysteine incorporation and related antioxidant proteins, Se status (as assessed by measurement of total serum Se, selenoprotein P (SePP) concentration and serum glutathione peroxidase (GPx3) activity) and prostate cancer risk in a European population with a Se status lower than that found in the USA. To achieve this aim, DNA samples from EPIC-Heidelberg, a prospective cohort study aiming to evaluate the association between dietary, lifestyle and metabolic factors and the risk of cancer, were genotyped and plasma samples analysed for plasma selenium status, selenoprotein P concentration and GPx activity. Previously, the samples had been analysed for six selenoprotein SNPs and rs1053040 in GPX1 was found to modulate the effect of serum Se on prostate cancer risk [13]. These six SNPs were not examined in the present study but instead the approach taken was a two-stage genotyping study: the first stage was an unbiased hypothesis-generating phase in which genotyping for 384 tagging-SNPs covering 72 Se-related genes (including selenoprotein genes, selenoprotein synthesis machinery, factors known to be influenced by selenoproteins, some related transcription factors and genes in linkage disequilibrium with these genes) was carried out in 94 advanced prostate cancer cases and an equal number of matched controls; in the second phase genotyping for a selected number of SNPs identified in the first phase was carried out in all 492 cases and controls.

Baseline characteristics of the study population have been presented previously [13]. Cases and controls were matched for age and were of comparable BMI. No significant differences were observed in mean serum concentration of Se or SePP or GPx3 activity between cases and controls [13].

Although previous work has suggested a possible inverse association between Se levels and risk of prostate cancer [4], studies of the influence of genetic variants in selenoprotein genes on prostate cancer risk or survival have been limited [13], [17], [18]. These earlier studies have provided evidence that variants in a combination of the SEPP1 and manganese superoxide dismutase (SOD2) genes affects disease risk [18], that rs1050450 in GPX1 affects the influence of Se status on risk [13] and that variants in SEP15 affect prostate cancer survival [17]. Increasingly, it is being realised that it is important to take an overall biological pathway approach to identify SNPs that show an association with a disease/disorder (e.g. [23], [24]). The present study used a two-phase genotyping approach to assess the influence of interaction of Se status and SNPs in genes across the selenium biological pathway on prostate cancer risk. The study extended earlier work by showing consistent significant interactions between serum markers of Se status and TXNRD1, TXNRD2 and SELK genotype with respect to risk of high-grade or advanced stage prostate cancer. As we used tagging-SNPs it is difficult to evaluate the functionality of these variants. Both TXNRD2 variants and the TXNRD1 variant studied are intronic and therefore the functional basis for the observed effects is unclear at present. In contrast, the SNP in SELK is in the promoter region and thus may influence gene expression through affecting promoter activity. A more detailed analysis of the genetic regions, combined with functional assays, could help in understanding the consequences of these SNPS or variants they are tagging.

The study involved analysis of samples from the EPIC-Heidelberg study and benefited from the availability of dietary and lifestyle data. However, the relatively small number of participants, particularly in the first phase of analysis, meant that the study was underpowered. Together with the lack of correction for multiple testing, this is a limitation that could lead to potential false positives that could have occurred by chance. However the strategy chosen here provides the opportunity to potentially identify new candidate functional SNPs and highlights the potential role of several selenoproteins in prostate function or prostate cancer etiology. Additionally, this approach reinforces previous observations [17] that the effects of some SNPs cannot be seen when Se status markers are not taken into account.

TR1 and TR2 proteins have major roles in regulation of redox signalling, whilst SelK has recently been reported to be an endoplasmic reticulum protein that is thought to play a role in the unfolded protein response and endoplasmic reticulum homeostasis [26], [27]. Both these biochemical processes are important in cell responses to metabolic challenges and are thought to be important in the control of cell proliferation and apoptosis. Therefore the observed effects of these SNPs are likely to reflect alterations in the ability of the prostate cells to respond to redox or inflammatory challenges. Interestingly, TR1 has been identified as one of four genes differentially expressed between androgen–dependent and independent growth of prostate cancer in mice [28]. Over-activation or dysfunction of thioredoxin reductases have been proposed to be involved in cancer etiology and prostate tumour progression [29], [30], [31]. As a result, thioredoxin reductases have been identified as anti-cancer targets and small molecules inhibitors of thioredoxin reductases (such as organoselenium compounds) are currently used in the treatment of prostate cancer [30], [31]. The observation that SNPs in both TXNRD1 and TXNRD2 genes are affecting prostate cancer risk supports the proposed role of the corresponding proteins in prostate function and/or tumour development. It would be interesting to determine if genetic variations in these two genes affect individual response to chemopreventive agents such as thioredoxin reductase inhibitors. In addition, SelK has been proposed to play a role in the regulation of Ca²⁺ flux [32] and changes in Ca²⁺ flux have been suggested to be involved in the progression to hormone-insensitive prostate cancer [33].

The association of SNPs in TXNRD1 and TXNRD2 with disease risk was only observed in advanced stage or high grade cancers and not in localized low-grade cases. This may reflect a role for these selenoproteins in the progression of prostate cancers rather that initiation, especially in view of the reported relationship between thioredoxin reductase activity and tumour aggressiveness [29]; alternatively, there may be different etiologies for the localized, low grade and the advanced, high grade cancers with different roles for the selenoproteins in the two disease situations. We hypothesize that sub-optimal Se status and altered activity of TR1, TR2 and SelK modify the ability of prostate cells to combat oxidative and inflammatory challenges and so affect their growth and tumour progression.

Selenoprotein expression in response to Se supplementation has been found to vary between individuals and some of these effects have been attributed to genetic polymorphisms in selenoproteins [34], [35], [36]. Synthesis of different selenoproteins responds differentially to sub-optimal Se intake [15], [37], [38] and since the protective effects of Se are thought to occur through selenoprotein biosynthesis one would expect potential genetic effects on prostate cancer risk to be modulated by Se status and vice versa. Since Se content of most foods depends on their geographical source Se intake is difficult to assess [1], but Se status can be assessed by measurement of blood biomarkers such as serum Se, plasma GPx or plasma SePP. SePP has been reported to be a better marker of status over a wider range of intake [12] than GPx3 but a recent systematic review concluded that more information is needed to evaluate their strengths and weaknesses as biomarkers of Se status [11]. Therefore, in this study we measured three markers of Se status and analysed them independently. There were significant interactions between Se status and TXNRD1, TXNRD2 and SELK genotype with respect to high-grade or advanced stage prostate cancer, so emphasising the importance of the combination of Se intake and genotype in determining prostate cancer risk. Our observations that effects of Se-related SNPs on prostate cancer risk were observed only in combination with Se status provide a likely explanation of why gene variants in selenoprotein genes have not been identified as risk factors in a recent genome-wide association study [25].

The lack of significant association of genotype for single SNPs in SEPP1 or SEP15 on prostate cancer risk is consistent with earlier reports that also found no association of specific SNPs in these genes with prostate cancer risk [17], [18]. However, in a nested case control study [17] variants in SEP15 were observed to be associated with prostate cancer mortality. In the present study we were unable to assess effects on mortality.

Large-scale Se supplementation trials in the USA have given contradictory outcomes with regards to evidence for a relationship between lower Se intake and increased risk of prostate cancer [5], [6]. This has been attributed to various factors, including higher baseline Se status in the SELECT study compared with the Nutritional Prevention of Cancer Trial [6]. The present study was carried out on European subjects in whom, on the basis of comparison of earlier populations [2], the mean Se status would be expected to be lower than in a US population. Indeed, as reported earlier [13] the mean plasma Se in control subjects within this study (87.7 µg/l) was lower than the baseline plasma Se in the recent SELECT trial [6]. In addition, there is evidence for familial associations of prostate cancer [39] and so it is also possible that the association between Se status and prostate cancer disease development may be modified by genetic variation in selenoproteins. Such genetic effects have been suggested to contribute to the differences in the outcomes of the Se supplementation trials [40].

In conclusion, this study shows a significant interaction between serum markers of Se status and TXNRD1, TXNRD2 and SELK genotype with respect to high-grade or advanced stage prostate cancer. This complements a study of the same cohort that focused on a small number of functional selenoprotein SNPs [13]. The earlier data showed that genotype for rs1050450 in GPX1 modified association of serum Se concentration with prostate cancer risk [13]. It also indicated that there was an association of borderline statistical between genotype for rs7579 in SEPP1 and prostate cancer risk [13]. Thus overall, the data from this EPIC-Heidelberg nested case-control study indicate that together Se status and GPX1, SEPP1, TXNRD1, TXNRD2, and SELK genotype significantly alter risk of high-grade or advanced stage prostate cancer in a population with suboptimal Se intake. Future studies should not only address functional effects of these variants in prostate tissue and but also focus on the larger studies needed to investigate the complex interplay of polymorphisms in different selenoproteins and Se status in prostate cancer development. This work also illustrates that approaches that take multiple SNPs within a metabolic pathway into account are particularly relevant to the study of SNP-nutrient interactions in relation to the risk for a complex disease as they take into consideration the different components of a biological pathway and nutritional biomarkers; indeed pathway enrichment methods to analyse data from genome-wide association studies have been developed [24].