Functional Variant of Manganese Superoxide Dismutase (SOD2 V16A) Polymorphism Is Associated with Prostate Cancer Risk in the Prostate, Lung, Colorectal, and Ovarian Cancer Study

Introduction

Superoxide dismutases (SODs) are a family of enzymes responsible for the detoxification of superoxide free radicals. Reactive oxygen species produced during chronic inflammation and other mechanisms involving oxidative stress may play an important role in prostate carcinogenesis. SODs are expressed in many human organs, including the prostate (1, 2). Thus, SOD genes are good candidates to evaluate genetic susceptibility for prostate cancer.

Among the three isoforms of SOD (SOD1, CuZn-SOD; SOD2, MnSOD; and SOD3, EC-SOD), SOD2 plays an important role as a primary mitochondria antioxidant enzyme and suppresses the growth of prostate cancer cells (1, 3-5). In this gene, several polymorphisms have been found, including Ex2+24T>C (V16A), which has been widely evaluated for association with various cancers, including prostate cancer (6-13).

The Ala variant at a mitochondrial targeting sequence of SOD2 allows more efficient SOD2 uptake into the mitochondrial matrix and generates more active SOD2 compared with the Val variant, suggesting that Ala/Ala homozygous subjects may have higher SOD2 activity (14-16). Contrary to this, two small epidemiologic studies on the association between SOD2 polymorphism and prostate cancer showed the Ala variant associated with increased risk of prostate cancer in poorly differentiated prostate cancer cases (17) or men with lower antioxidant intakes (18).

Two other isoforms of SOD with Cu and Zn in their catalytic center (CuZn SODs) are localized in intracellular cytoplasmic compartments (SOD1) and extracellular elements (SOD3), respectively. SOD1 polymorphisms have not been found to be associated with breast cancer risk (9, 10). SOD3 Ex3-631G (231G) allele has been reported to increase the serum level of SOD3 (19, 20); however, it has not been evaluated for the association with cancer risk.

We evaluated the association of prostate cancer with genetic polymorphisms in the three main isoforms of SOD, and explored potential risk modification by intake of antioxidants, in a large case-control study nested in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial.

Materials and Methods

Study Setting

This nested case-control study was conducted within the screening arm of the PLCO Trial, which was designed to evaluate the effectiveness of screening intervention for prostate, lung, colorectal, and ovarian cancer and to investigate etiologic factors and early markers of cancer (21, 22). Participants in the PLCO Trial, ages 55 to 74 years, were recruited at 10 centers in the United States (Birmingham, Alabama; Denver, Colorado; Detroit, Michigan; Honolulu, Hawaii; Marshfield, Wisconsin; Minneapolis, Minnesota; Pittsburgh, Pennsylvania; Salt Lake City, Utah; St. Louis, Missouri; and Washington, District of Columbia) between September 1993 and June 2001.

Men randomized to the screening arm of the trial were offered prostate cancer screening by serum prostate-specific antigen, at entry and annually for 5 years, and digital rectal examination, at entry and annually for 3 years. If the prostate-specific antigen test result was ≥4 ng/mL or digital rectal examination was suspicious for prostate cancer, men were referred to their medical care providers for prostate cancer diagnostic evaluation. In addition, annual follow-up for recent diagnosis of cancer was carried out by mailed questionnaires and through periodic search of the National Death Index. All medical and pathologic records related to diagnosis were obtained for those who might be prostate cancer case. Furthermore, death certificates and supporting medical/pathologic records were collected. All data related to diagnosis of cancer and death were abstracted by trained medical record specialists. Blood samples were drawn from screening arm participants at each of the screening visits. Participants provided written informed consent. The trial was approved by the institutional review boards of the U.S. National Cancer Institute and the 10 study centers.

Study Population

Men randomized to the screening arm were eligible for this nested case-control study if they had at least one valid screening for prostate cancer (prostate-specific antigen and/or digital rectal examination) before October 1, 2001 (the censor date for this analysis), completed the baseline risk factor questionnaire, provided a blood sample, and signed the informed consent for studies on cancer (n = 26,975). All men were followed from their initial valid prostate cancer screen (prostate-specific antigen and/or digital rectal examination) to first occurrence of prostate cancer, loss to follow-up, death, or October 1, 2001, whichever came first. Cases were defined as men diagnosed with adenocarcinoma of the prostate. We excluded all cases that were diagnosed within the first year after the initial prostate cancer screen (prostate-specific antigen and/or digital rectal examination). Staging procedures corresponded to the tumor-node-metastasis stage of disease classification. Cases were classified as clinically significant (n = 562) if they were stages III or IV or Gleason score ≥7. The eligible group included 1,320 prostate cancer cases (1,213 non-Hispanic Caucasians and 107 African-Americans). For comparison, we selected 1,842 controls (1,433 non-Hispanic Caucasians and 409 African-Americans) using risk-set sampling frequency matched to cases by age (55-59, 60-64, 65-69, and 70-74), ethnicity (case-control ratio of 1.2:1 for Caucasians and 4:1 for African-Americans), time since initial screening (1-year time windows), and year of blood draw (1-year calendar periods; ref. 23).

Assessment of Questionnaire-Based Covariates

At enrollment, all participants were asked to complete a questionnaire, including age, ethnicity, education, occupation, current and past smoking behavior, history of cancer and other diseases, use of selected drugs, recent history of screening exams, and prostate-related health factors. Usual dietary intake over the 12 months before enrollment was assessed with a 137-item food frequency questionnaire, including an additional 14 questions about intake of vitamin and mineral supplements and 10 questions on meat cooking practice (PLCO Cancer Screening Trial).⁸ Daily dietary nutrient intake was calculated by multiplying the daily frequency of each consumed food item by the nutrient value of the sex-specific portion size (24) using the nutrient database from the U.S. Department of Agriculture (25). Total vitamin and mineral intake was calculated by adding dietary and supplemental intake.

Serum Selenium Analysis

Serum selenium concentrations were determined using an inductively coupled plasma mass spectrometry method (26). Serum for selenium analysis was available for 724 (92.6%) cases and 879 (94.7%) controls. Cases and their matched controls were analyzed in the same batch. Quality control samples were monitored throughout the analysis. The coefficient of variation estimated from 181 duplicates was 9.4%.

SOD1/SOD2/SOD3 Genotyping

Genotype analysis was done at the National Cancer Institute Core Genotyping Facility.⁹ All Taqman assays (Applied Biosystems, Inc.) were optimized on the ABI 7900 HT detection system with 100% concordance with sequence analysis of 102 individuals listed on the SNP500Cancer Web site (27).¹⁰ Six SNPs in three genes were genotyped: SOD1 IVS3-251A>G (rs2070424), SOD2 Ex2+24T>C (V16A; rs1799725), and SOD3 IVS1+186C>T (rs699473), Ex3-631C>G (R231G; rs1799895), Ex3-516C>T (rs2853796), and Ex3-489C>T (rs2855262). These SNPs were selected based on functional significance, the results of previous association studies with cancer, and minor allele frequency criteria (>5%; refs. 6-8, 11, 12, 14-16, 19, 20, 27).

The Hardy-Weinberg equilibrium for each SNP was tested with Pearson χ² or exact test if any of the cell counts was small. The genotype distributions of SOD3 Ex3-516C>T and Ex3-489C>T diverted from Hardy-Weinberg equilibrium in African-American controls (P = 0.02 and 0.003, respectively). However, no deviations from Hardy-Weinberg equilibrium were observed in the Caucasian controls (P > 0.05), and the concordance rates for quality control samples (n = 253), which were replicates from 48 study subjects interspersed throughout study samples, were >99% for the two assays.

Statistical Analysis

We used conditional logistic regression models to estimate odds ratios (OR) and 95% confidence intervals (95% CI) of prostate cancer. All P values are two sided. The analyses were conditioned on the matching factors (age, time since initial screening, and year of blood draw). Genotype data were analyzed with the homozygote of the common allele as the reference group. Variants of homozygotes and heterozygotes were combined to evaluate the dominant effect. For each SNP, trend tests were conducted by assigning the ordinal values 1, 2, and 3 to homozygous wild-type, heterozygous, and homozygous variant genotypes, respectively, and by adding these scores as a continuous variable in logistic regression model.

To explore potential effect modification by antioxidants intake, we did stratified analyses and evaluated multiplicative interaction by creating product terms. Statistical significance of multiplicative interactions was evaluated by comparison of the log likelihood statistics between the main effect model and the joint effects model.

Haplotype analyses for SOD3 were conducted using the haplo.stats package¹¹ in the R program (version 2.2.1),¹² which uses an expectation-maximization algorithm to estimate haplotypes from genotype data (28). The Ex3-631C>G (R231G) polymorphism was excluded because of low variant allele frequency (G: <1.5%). Haplotypes were estimated separately for Caucasians and African-Americans excluding subjects missing all the genotype data. Haplotypes were estimated for persons missing one to two genotypes using an extension of the expectation-maximization algorithm. Each haplotype pair was weighted by its probability, rather than assigning a most likely haplotype to an individual, and the generalized linear model implemented in haplo.stats was used to estimate the effect of individual haplotypes by fitting an additive model, adjusting for age, time to diagnosis, and year of blood draw. The overall difference in haplotype frequencies between cases and controls was assessed by race using a global score test, adjusting for age, time to diagnosis, and year of blood draw.

Results

The characteristics of 1,320 cases and 1,842 controls are shown in Table 1 . Briefly, age distribution was similar between cases and controls: mean age was 64.8 (±5.0) for cases and 64.5 (±5.0) for controls. The proportion of African-Americans was higher in controls than in cases due to oversampling (4:1) of African-American controls. Mean body mass index was marginally different and percentages with family history of prostate cancer were significantly different (P < 0.001) between cases and controls; smoking and alcohol consumption were not associated with case-control status.

The Ala variant of SOD2 Ex2+24T>C (V16A) was associated with increased risk of prostate cancer in Caucasians (Val/Ala versus Val/Val: OR, 1.17; 95% CI, 0.97-1.42; Ala/Ala versus Val/Val: OR, 1.28; 95% CI, 1.03-1.60; P_trend = 0.03); number of African-American carriers with the Ala/Ala genotype was small, making inferences difficult. No other significant associations were noted in Caucasians or African-Americans (Table 2 ). When stratified by the level of dietary and supplemental intake, significant trends were noted in men with dietary and supplemental intake of vitamin E below the median (31.2 IU/d; Val/Ala versus Val/Val: OR, 1.58; 95% CI, 1.17-2.12; Ala/Ala versus Val/Val: OR, 1.56; 95% CI, 1.11-2.20; P_trend = 0.01; Table 3 ). Further stratification by quartiles of dietary and supplemental vitamin E intake (Q1 = 13.3 IU/d, Q2 = 31.2 IU/d, and Q3 = 222 IU/d) showed risks of prostate cancer tended to be increased among SOD2 Ala allele carriers, except at the highest quartile of vitamin E intake (>222 IU/d; P_interaction = 0.06, Q1-Q3 versus Q4; Fig. 1 ). Risks tended also to follow this pattern for clinically significant disease (stages III/IV or Gleason score ≥7; P_interaction = 0.02); similar results were observed when advanced stage and Gleason score were evaluated separately. Among those with lower intake of vitamin E (Q1-Q3: <222 IU/d), the association between Ala-containing genotypes and prostate cancer was stronger for smokers (OR, 1.44; 95% CI, 1.10-1.90) compared with nonsmokers (OR, 1.17; 95% CI, 0.78-1.75; Table 4 ). Interaction test result for SOD2 genotype and vitamin E was marginally significant among smokers (P_interaction = 0.08) and not significant among nonsmokers (P_interaction = 0.44). SOD2 Ala–associated risks did not vary significantly with respect to dietary intake of β-carotene or lycopene or serum selenium level.

No significant associations with prostate cancer were observed for polymorphic variants in SOD3 or SOD1 (Table 2). Haplotype frequencies of SOD3 were also not significantly different between cases and controls in either Caucasians or African-Americans (P_omnibus = 0.60 and 0.20, respectively; data not shown).

Discussion

Our results suggest that the Ala variant of SOD2 Ex2+24T>C (V16A) was associated with a moderately increased risk of prostate cancer in Caucasians, particularly among men with lower intake of dietary and supplemental vitamin E. No significant associations with prostate cancer were observed for polymorphic variants in SOD3 or SOD1. Similar to our study, men homozygous for the SOD2 Ala allele had a 70% increase in risk over men homozygous for the Val allele in Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study conducted for 197 cases and 190 controls (17), whereas no overall association was found in the Physician's Health Study conducted for 567 cases and 764 controls (18).

In our study, the elevation of prostate cancer risk associated with the SOD2 Ala allele was observed particularly for smokers with lower vitamin E intake. We previously reported, in the PLCO Trial, that greater vitamin E intake was associated with lower prostate cancer risk, only in smokers (29), as also reported by several other groups (30, 31). The current report suggests that SOD2 may be involved in this relationship. Similar to our findings, others reported the homozygous Ala genotype associated with a 2.5-fold increased risk for prostate cancer among men with low antioxidant status (18) and with premenopausal breast cancer in women with low dietary antioxidant intake (6, 8).

SOD2 plays a pivotal role in protecting cells from reactive oxygen species–induced oxidative damage and is a known tumor suppressor in prostate cancer cells (1, 3-5). The Ala variant of SOD2 allows more efficient SOD2 import into the mitochondrial matrix and generates more active SOD2 compared with the Val variant (14-16). Thus, it is hypothesized that the higher activity (Ala) variant suppresses prostate carcinogenesis; however, our results and previous association studies on prostate cancer contradict this hypothesis (17, 18).

Alternatively, increased levels of hydrogen peroxide (H₂O₂) are found in prostate cancer cells (32) and have been related to overexpression of SOD2 (33). H₂O₂ is a major intracellular oxidant involved in H₂O₂-induced DNA damage in prostate cancer (34) and in induction of genes for prostate carcinogenesis (33, 35). Thus, the SOD2 Ala allele might increase the risk of prostate cancer by producing excessive H₂O₂.

Our study was limited in that only one SNP was evaluated in SOD1 or SOD2 and the haplotype analysis for SOD3 was not comprehensive. However, SOD1 is a small gene and alleles in SOD1 show strong linkage disequilibrium in Caucasians (9). In addition, SOD2 Ex2+24T>C (V16A) itself is located in mitochondrial targeting sequence and has experimental evidences of functional significance in vivo (16) as well and in vitro (14, 15). Another limitation is insufficient statistical power in subgroup analysis for African-Americans. Although we presented data for African-Americans, sample size was small and conclusions for this group are limited.

Our study, the largest to evaluate the role of genetic polymorphisms in SOD genes and prostate cancer risk, indicates that the Ala variant of SOD2 Ex2+24T>C (V16A) is associated with increased risk for prostate cancer, particularly among smokers and men with lower intake of dietary and supplemental vitamin E.