Interaction between SNPs in selenoprotein P and mitochondrial superoxide dismutase determines prostate cancer risk

Abstract

INTRODUCTION

Selenium, an essential nutrient, may reduce the incidence and/or progression of prostate cancer particularly in men with the relatively-low baseline selenium status commonly found in Europe [1-4]. The potential anti-cancer effects of selenium may be exerted through a number of parallel mechanisms some of which involve the selenoproteins [1]. Indeed recent evidence suggests an important role for selenoproteins in cancer [1, 5], specifically in prostate cancer [6].

Selenoprotein P contains at least 40% of the total selenium in plasma [7]. Deletion of the gene for selenoprotein P in mouse models alters the distribution of selenium in body tissues suggesting that selenoprotein P is required for selenium transport [8, 9]. While the human selenoprotein P gene (SEPP1) is abundantly expressed in normal colon mucosa, there is a significant reduction or loss of SEPP1 mRNA expression in colon cancers [10]. Expression of SEPP1 is also dramatically reduced in a subset of human prostate tumors, mouse tumors and in the androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cell lines [11]. Homozygosity for the Ala234 allele of the SEPP1 Ala234Thr SNP (rs3877899) is associated with a lower concentration of plasma selenoprotein P in men, affecting the concentration and/or activity of other selenoproteins, notably of thioredoxin reductase 1 (TR1) and some of the antioxidant glutathione peroxidases (GPx) [12]. Thus it is conceivable that SEPP1 genotype may affect prostate cancer risk.

Selenium status is linked to the effect of another polymorphism on prostate cancer risk i.e. that of mitochondrial superoxide dismutase (SOD2 or MnSOD) [13], the major detoxifying enzyme in the mitochondrion. This enzyme dismutes superoxide to H₂O₂, which must itself then be detoxified to water by GPx [13]. A well-characterized polymorphism in SOD2 results in the substitution of alanine (Ala) for valine (Val) at codon 16 (rs4880) and a higher activity of the Ala16 mitochondrial enzyme [14]. Among US men homozygous for Ala16, those whose plasma selenium was in the top quartile had a relative risk (RR) of prostate cancer of 0.3 (95% CI 0.2 to 0.7) and of clinically aggressive prostate cancer of 0.2 (95% CI 0.1 to 0.5) when compared with those whose plasma selenium was in the bottom quartile [13].The dependence on selenium status of this genotype effect on prostate cancer risk may relate to the requirement for adequate GPx to remove the extra H₂O₂ formed in Ala16 homozygotes [13].

We investigated the relationship of these two putatively-functional polymorphisms to prostate cancer risk in the large CAPS (CAncer Prostate in Sweden) study [15]. We hypothesised that in a low selenium environment, as in Sweden, men who have SNP alleles that both reduce their ability to make functional selenoprotein P (SEPP1 Ala234 homozygotes) and increase their production of H₂O₂ (SOD2 Ala16) thereby increasing their requirement for GPx, would have a greater risk of prostate cancer than men who do not have these alleles.

SUBJECTS AND METHODS

RESULTS

Interaction between SOD2 and SEPP1 polymorphisms

Men homozygous for the SEPP1 Ala234 allele, who were also SOD2 Ala16+, were at 43% greater risk of prostate cancer than SOD2 Val16 homozygotes (adjusted OR 1.43; 95% CI 1.17 to 1.76; P = 0.0005; Table 2, Fig. 1). By contrast, there was no association between SOD2 genotype and cancer risk in SEPP1 Thr234+ men. This interaction between the two SNPs in determining risk of prostate cancer had a borderline statistically significant P value of 0.05.

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Figure 1

The interaction between SOD2 Val16Ala and SEPP1 Ala234Thr SNPs and risk of: A all prostate cancer; B aggressive prostate cancer; C prostate cancer in never-smokers and D prostate cancer in ever-smokers, in the CAPS study. OR = odds ratio, adjusted for age and geographical location. R indicates the referent group (foreground column) for each comparison with the alternative SOD2 genotype group (background column).

Table 2

The interaction between SOD2 Val16Ala and SEPP1 Ala234Thr SNPs and risk of all prostate cancer, non-aggressive prostate cancer and aggressive prostate cancer in the CAPS study.

	Controls	All prostate cancer			Non-aggressive prostate cancer			Aggressive prostate cancer
Genotype	n	n	OR (95% CI)*	P	n	OR (95% CI)*	P	n	OR (95% CI)*	P
SEPP1 Codon 234 Ala/Ala	816	1360			854			506
SOD2 Val/Val	224	286	Referent	1	189	Referent	1	97	Referent	1
SOD2 Ala/Val + Ala/Ala	592	1074	1.43 (1.17 to 1.76)	0.0005	665	1.36 (1.09 to 1.71)	0.0073	409	1.60 (1.22 to 2.09)	0.0007
SEPP1 Codon 234 Ala/Thr + Thr/Thr	638	1009			631			378
SOD2 Val/Val	161	246	Referent	1	147	Referent	1	99	Referent	1
SOD2 Ala/Val + Ala/Ala	477	763	1.06 (0.84 to 1.34)	0.62	484	1.14 (0.87 to 1.48)	0.35	279	0.95 (0.71 to 1.28)	0.75
P for interaction			0.051			0.278			0.012

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^*odds ratio adjusted for age and geographical location, 95% confidence interval

n = Number of subjects

In aggressive prostate cancer, the interaction between the SNPs was stronger (P = 0.01) with SEPP1 Ala234 homozygotes who were also SOD2 Ala16+ having a 60% increased risk of aggressive disease compared to SOD2 Val16 homozygotes (adjusted OR 1.60; 95% CI 1.22, 2.09; P = 0.0007; Table 2, Fig. 1), whereas there was no association with SOD2 genotype in the SEPP1 Thr234+ men. Although, the association with SNP genotypes in non-aggressive disease might appear significant, the interaction between the two SNPs was far from statistical significance as demonstrated by the broad overlap between the odds ratio confidence intervals in the two SEPP1 genotype groups (Table 2).

DISCUSSION

The mean plasma selenium of 76.0 ± 17.2 μg/L in control samples confirms the relatively low selenium intake and status in the Swedish population (cf mean US value of 125 μg/L determined in the Third National Health and Nutrition Examination Survey [19]). This value is less than the 92 μg/L required for maximal plasma GPx activity [20] and considerably less than the plasma concentration required for full expression of SEPP1 [7, 21], demonstrating that the study population has a selenium intake inadequate for optimal selenoprotein synthesis and/or activity. This may be relevant to prostate cancer risk since low selenoprotein production led to higher-grade lesions and aggressive disease in a transgenic mouse model of prostate cancer [6]. We reasoned that in a population with relatively-low selenium status, inter-individual variation in selenium requirement, as determined by selenoprotein genotype, might have a greater effect on the risk of prostate cancer than in a selenium-replete population.

Despite our rationale, we found no effect of SEPP1 genotype per se on the risk of prostate cancer or on non-aggressive or aggressive prostate cancer. A similar null-effect of this genotype was found in colorectal cancer [10]. We did, however, find an effect of genotype in a pathway associated with selenoprotein function: although SOD2 is not a selenoprotein, the product of its activity, H₂O₂, is a substrate for GPx. The Val16Ala SNP in SOD2 has been shown to alter the secondary structure of the mitochondrial import sequence of the superoxide dismutase protein such that the Ala16 variant is imported more efficiently into the mitochondrial matrix, resulting in higher enzyme activity [22]. Individuals with at least one SOD2 Ala16 allele (Ala16+) therefore generate more active superoxide dismutase (and therefore more H₂O₂) than those homozygous for the Val16 variant. In our study, SOD2 Ala16+ men were at a 19% increased risk of prostate cancer compared to Val16 homozygotes (Table 1). The mitochondrion contains little or no catalase and so is entirely dependent on the activity of GPx to remove H₂O₂ [23], though of course H₂O₂ is sufficiently long-lived to diffuse out of the mitochondrion.

H₂O₂ promotes prostate cancer cell proliferation and migration [24, 25], and induces matrix metalloproteinases required for tumor invasion [26, 27]. For instance, H₂O₂ levels rose in cell-lines of the LNCaP series as tumorigenic and metastatic potential increased [24]. Furthermore, addition of ebselen, a GPx mimetic, to the assay completely abolished the chemiluminescence attributable to H₂O₂ [24]. At low selenium concentration where there is insufficient GPx activity to remove H₂O₂, the SOD2 16Ala allele would therefore be expected to have a deleterious effect owing to the higher H₂O₂ production associated with that allele variant. Our population has a mean plasma selenium concentration well below the level required to fully optimise GPx (as plasma GPx) [20] making our observation consistent with predictions.

Furthermore, the mean selenium concentration in our study was below the bottom of the range of plasma selenium (84-131 μg/L) in the study in which Li et al [13] reported an association between SOD2 Ala16Val genotype and prostate cancer risk when subjects were divided according to quartile of selenium status. In that study, high selenium status was advantageous for SOD2 Ala16 homozygotes as the combination of high SOD2 activity in the mitochondrion, together with high selenium/GPx, enabled efficient removal of both reactive oxygen species, superoxide and H₂O₂. By contrast, Ala16 homozygotes were at increased risk, particularly of aggressive prostate cancer, when their selenium status was in the bottom quartile [13]. While we saw an overall effect of SOD2 Ala16Val genotype in our low-selenium population, Li et al [13] observed no overall effect, perhaps because the range of selenium status in their population encompassed both positive and negative effects of SOD2 genotype on prostate cancer risk leading to a null effect overall. Our findings might help explain why the SOD2 Ala16Val risk allele appears to vary from study to study in a number of cancers and illustrates a gene-nutrient interaction where the effect of genotype on risk reverses with change in nutrient status of the population.

Results from the ATBC study may further illustrate this point. Finnish smokers who were SOD2 Ala16 homozygotes had a 70% increased risk of prostate cancer when compared to Val homozygotes [28]. This is consistent with the findings of Li et al in their lowest selenium quartile where the SOD Ala16 homozygotes had an increased risk of prostate cancer [13] and suggests that men from the ATBC cohort must have had a similar selenium status to the bottom quartile of US men. Although at first sight this might seem surprising since the cohort was recruited after the introduction of selenium-enriched fertilizers in Finland, in fact there are data to show that the ATBC study did indeed include men with very low Se status [29]. When the use of selenized fertilisers was implemented in 1984, mean plasma selenium in Finland was 70 μg/L. However, selenium status did not peak until 1990, reaching a maximum of 120 μg/L (cf mean US value 125 μg/L [19]), before declining to 90 μg/L in 1999 [30]. Thus the mean selenium status of this Finnish cohort, recruited from 1985-88 and followed up until death or 1993, would have been considerably lower than that of the US men and would certainly have been lower during the time this slow-growing cancer was developing, prior to the initiation of the selenized fertilizer program.

We found evidence for a gene-gene interaction between the Ala234Thr polymorphism in the Se transport protein, SEPP1, and the SOD2 Val16Ala polymorphism. Individuals homozygous for SEPP1 Ala234, SOD2 Ala16+ were 43% more likely to have prostate cancer compared to Val16 homozygotes (Table 2); this interaction was stronger in aggressive disease resulting in a 60% increased risk. Prospective cohort studies have shown a stronger association between selenium status and risk of aggressive than localized disease [31-35].

Of relevance to our findings, Méplan et al recently found that male SEPP1 Ala234 homozygotes had lower plasma selenoprotein P than heterozygotes, with an associated trend for reduced activity or protein concentration of GPx1 (cytosolic GPx) and GPx4 (phospholipid GPx) [12]. The authors suggest that the Ala234Thr polymorphism affects the stability of selenoprotein P, possibly through a post-translational modification, affecting protein levels only when selenium intake is suboptimal. Thus genotype-dependent differences in plasma selenoprotein P concentration were only observed under relatively low selenium conditions and disappeared after supplementation with selenium [12]. Our observations in a population of relatively-low selenium status are consistent with those findings.

Because there is strong evidence that selenoprotein P plays a critical role in delivery of hepatic selenium to other tissues [8, 9, 36], we hypothesise that men homozygous for the SEPP1 Ala234 allele have reduced availability of selenium for the production of GPx in the prostate. This would impede the ability of the prostate to remove H₂O₂ and would therefore increase the risk of prostate cancer [24, 25] (Fig. 2). In our study, this polymorphism only affected prostate cancer risk in conjunction with the SOD2 Ala16 allele, which, by allowing more efficient transport of superoxide dismutase into the mitochondrion, caused increased production of H₂O₂ that became detrimental in the face of low selenium. The gene-gene interaction between the SEPP1 Ala234Thr and SOD2 Val16Ala polymorphisms was also apparent in current or ex-smokers who had a two-fold increased risk of prostate cancer compared with SOD2 Val16 homozygotes (Table 4). As we have postulated that the mechanism by which these polymorphisms have their combined effect is oxidative-stress related, the greater strength of the interaction in smokers than in the study as a whole, despite smaller numbers, might be explained by an exacerbation of oxidative stress in ever-smokers though it may equally well be related to the lower selenium status seen in smokers [37, 38]. In fact we did not observe lower selenium status among smokers in our study but this may have been due to the small number (n=169) of plasma selenium measurements made.

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Figure 2

Postulated mitochondrial mechanism for the interaction between SEPP1 and SOD2 polymorphisms and the risk of prostate cancer. Possession of a SOD2 Ala16 allele promotes higher import of mitochondrial superoxide dismutase to the mitochondrion resulting in higher activity and greater production of H₂O₂ than with the SOD2 Val16 variant. The availability of selenium for the production of GPx in the prostate, already low in the Swedish population, is further reduced in men homozygous for the SEPP1 Ala 234 allele, reducing the ability of the prostate to protect against excess hydrogen peroxide (promoter of cell proliferation and migration[24, 25]) and cancer.

Our study has limitations in that HapMap‡ shows considerable linkage disequilibrium (LD) at both these gene loci so we cannot be sure that these polymorphisms are the only functional SNPs affecting risk of prostate cancer in the SOD2 and SEPP1 genes. However, given the published studies implying functional consequences of the amino-acid changes, it would seem plausible that they have some function in this context. Both of these SNPs were included in the recent genome-wide screen of a subset of our case-control study (498 aggressive cases and 494 controls) and results are consistent with the present data on a larger sample [39]. Other recent genome-wide SNP screens in prostate cancer have not implicated either of these genes [40-42], but the UK study did find an associated SNP (rs9364554) in the SLC22A3 gene [40] on chromosome 6, just over 700kb telomeric of SOD2. Analysis of HapMap CEPH data reveals no LD between this SNP and SOD2 Val16Ala, suggesting that the SOD2 SNP is independently associated with prostate cancer risk and is not acting as a marker for SLC22A3 association.

If confirmed, the evidence presented here would allow identification of individuals who would particularly benefit from selenium supplementation to prevent prostate cancer. Among the Swedish men in our study, 41% had the high risk genotype combination (homozygosity for SEPP1 Ala 234 and possession of an SOD2 Ala16 allele). Similar allele frequencies have been identified in other populations of European descent † [12, 13]. We have observed the combined effect of the SOD2 Val16Ala and SEPP1 Ala234Thr SNPs under conditions of limited selenium availability. Such an effect can probably not be observed in areas of higher selenium status such as North America where plentiful selenium supply can probably obviate the disadvantage of homozygosity for SEPP1 234 Ala except for those with the lowest intakes [13]. Optimising the selenium intake of individuals homozygous for SEPP1 Ala234 will improve their ability to supply selenium via selenoprotein P for GPx synthesis and H₂O₂ removal [12]. Confirmation of our findings in other populations with low selenium intake should be a high priority. So far, few genetic tests have had the potential to ameliorate risk by identifying the need for a simple, inexpensive, nutritional supplement.

Acknowledgments

Abbreviations

Footnotes

References