A Common Prostate Cancer Risk Variant 5′ of Microseminoprotein-β (MSMB) Is a Strong Predictor of Circulating β-Microseminoprotein (MSP) Levels in Multiple Populations

β-Microseminoprotein (MSP) has been implicated as a biomarker of prostate cancer risk, detection, and prognosis (1, 2). MSP, also called prostate secretory protein of 94 amino acids, is one of the most highly secreted proteins by the prostate gland (3). Aside from an abundant occurrence of MSMB transcripts and MSP protein in the prostate, abundant expression has also been found in the trachea and bronchi in the respiratory tract, and in the gastric mucosa, which may help to explain why circulating levels are quite comparable in women and in men <40 years old (4-7). MSP may have a physiologic role in fertility because it has been shown to bind sperm and inhibit the acrosome reaction (8). In the prostate, MSP expression has been observed to be higher in benign than in cancerous tissue (9). Serum MSP levels have been found to be lower in men with aggressive prostate cancer (1) as well as in men with a prostate cancer recurrence after radical prostatectomy (2, 10). Studies in vivo and in vitro have also implicated MSP as having a functional role in regulating cellular growth in the prostate through apoptosis (11).

Genome-wide association studies of prostate cancer have identified a common risk variant (rs10993994) in the 5′ region of MSMB on chromosome 10q11.2 (the gene that encodes MSP; refs. 12, 13). We recently replicated the association of this variant with prostate cancer risk in a multiethnic population (14). The variant, rs10993994, is located 57 bp upstream of the first exon of MSMB with the C allele (hypothesized low-risk allele) found to preferentially bind the CREB transcription factor and to be associated with higher MSMB gene expression in tumor cell lines (15, 16). A recent study among Chinese men, in which 60 of 251 cases and 30 of 258 controls were evaluated for the potential association between MSP levels and MSMB genotype, reported serum MSP levels to be lower in those who carried the CT or TT genotype than in those with the CC genotype among the 60 cases (17). These genetic data, together with the clinical and laboratory observations, are consistent with the hypothesis that lower MSMB expression is associated with prostate cancer development.

In an attempt to clarify the biological mechanism underlying the association of genetic variation at the MSMB locus and prostate cancer risk, we examined the association between rs10993994 genotype and MSP levels in the blood plasma from prostate cancer–free men from four racial/ethnic populations (European Americans, African Americans, Latinos, and Japanese Americans). We also examined known and putative risk factors for prostate cancer as potential contributors to interindividual variability of circulating levels of MSP in the study population.

This study consisted of male participants of the Multiethnic Cohort. The Multiethnic Cohort includes 96,810 men and 118,441 women, and is comprised mainly of African Americans, Japanese Americans, Latinos, Native Hawaiians, and European Americans (18). Between 1993 and 1996, adults between 45 and 75 years old were enrolled by completing a 26-page, self-administered questionnaire asking detailed information about demographic factors, personal behaviors, dietary habits, and history of prior medical conditions. Potential cohort members were identified through Department of Motor Vehicles drivers' license files, voter registration files, and Health Care Financing Administration data files. Between 1995 and 2006, blood specimens were collected from ∼67,000 Multiethnic Cohort participants for genetic and biomarker analyses.

Blood plasma MSP and prostate-specific antigen (PSA) levels, and rs10993994 genotype were measured in 500 men from four racial/ethnic groups (n = 125 each of European Americans, African Americans, Latinos, and Japanese Americans). Blood samples of the men included in this study were collected between 1994 and 2004, and were cancer-free, as determined through linkage of the cohort with Surveillance Epidemiology and End Results cancer registries in California and Hawaii, as of January 1, 2009. Men were randomly selected from the control group of the nested prostate cancer case-control study in the Multiethnic Cohort (19). We limited the study to men in the age range of 50 to 69 years old at the time of blood draw because this is the age when men are typically PSA screened for prostate cancer (20). We also limited the study to men with a self-reported body mass index (BMI; kg/m²) between 19 and 35 to avoid subjects with extremely high or low BMI levels in which the effects on MSP are not known. Four hundred twelve (82%) of the men had fasted ≥8 hours before blood collection. The Institutional Review Boards at the University of Southern California and the University of Hawaii approved the study protocol.

Archived EDTA anticoagulated blood plasma samples (stored frozen for 3-14 y at −80°C after initial processing within 4 hours from venipuncture) were retrieved and shipped frozen on dry ice to Malmö, Sweden in fall of 2009. Analyses of free and total PSA, and MSP were done blinded to ethnicity or genotyping data in Dr. Lilja's laboratory at the Wallenberg Research Laboratories (Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden) during 2009 to 2010. Production and purification of the polyclonal rabbit anti-MSP antibody and seminal plasma MSP have been described elsewhere (21). Biotinylation and Europium labeling of the anti-MSP antibody were done as previously described (22). Streptavidin-coated microtiter plates were from Kaivogen Oy. Assay buffer and wash solution have been described previously (21).

The MSP immunoassay of plasma MSP levels, which have previously been shown to not be significantly different from serum MSP levels (4), was done with the AutoDelfia 1235 automatic immunoassay system (Perkin-Elmer Life Sciences). First, 150 ng of the biotinylated MSP antibody was attached to the streptavidin-coated wells in 100 μL of assay buffer during a 30-minute incubation. After two washes, 100 μL of assay buffer and 10 μL of standard or sample material were added to the wells, incubated 1 hour and washed twice. The Eu-labelled anti-MSP antibody (50 ng) was added in 200 μL of assay buffer. After 1-hour incubation, the wells were washed six times before adding 200 μL of the Delfia enhancement solution (Perkin-Elmer Life Sciences), followed by incubation for 5 minutes at room temperature with shaking before the time-resolved measurement of the Eu fluorescence. To measure free and total PSA, we used the dual-label DELFIA Prostatus total/free PSA assay (Perkin-Elmer; ref. 23), which is calibrated against WHO 96/670 (PSA-WHO) and WHO 68/668 (free PSA-WHO) standards. Samples were run in eight batches. Mean coefficients of variation of 7.4%, 11.8%, and 7.8% were observed for MSP, total PSA, and free PSA, respectively, among 20 blinded duplicate samples (5 per ethnic group).

Genotyping of rs10993994 and other genome-wide association study–validated risk alleles for prostate rcance was done with the use of the Taqman allelic discrimination assay, as previously described (14, 19, 24). The DNA for genotyping was extracted from buffy coat samples with the use of a QIAamp 96 DNA blood kit (Qiagen). Of the ∼5% duplicate samples used to assess genotyping reproducibility, there was 100% concordance for rs10993994.

MSP and PSA levels were log transformed to better normalize the distributions, and mean levels are presented as least squares geometric means. Generalized linear models were used to examine the association between rs10993994 and MSP, adjusted for age (continuous), BMI (continuous), laboratory batch, and race/ethnicity (pooled analysis). Laboratory batch was not significantly associated with MSP levels. We also adjusted for PSA levels because PSA was found to be modestly correlated with MSP levels. Adjustment for either total, free PSA (ng/mL), or percent free PSA provided similar results. The least squares geometric mean MSP levels and the percent change relative to the wild-type (i.e., low risk) rs10993994 genotype (CC) was calculated in ethnic-specific analyses. The effect of global European ancestry on the association between MSP levels and rs10993994 was examined in the admixed racial/ethnic populations (African Americans and Latinos) with the use of ancestry estimates from a previous study (19). Fasting time before blood collection was not included in the analysis because it was not significantly associated with MSP levels and had no effect on the association between rs10993994 and MSP.

Because the determinants of MSP levels are largely unknown, we also examined other known and suspected risk factors for prostate cancer in relation to MSP levels, including demographic factors, dietary factors, and established common risk variants from genome-wide association studies. More specifically, we examined age at the time of blood draw, BMI (kg/m²), weight (kg), height (cm), total PSA (ng/mL), free PSA (ng/mL), percent free PSA, physical activity (hours of vigorous work and exercise), and intake of saturated fat (percentage of calories from saturated fat), red meat (calorie-adjusted intake per day; g/kcal/d), lycopene (calorie-adjusted intake per day; 100 mcg/kcal/d), calcium (calorie-adjusted intake per day; mg/kcal/d), vitamin D (calorie-adjusted intake per day; IU/kcal/d), and alcohol (g/d). We tested the association between an increase of 1 unit of each factor and the percent change in geometric mean MSP levels. Age, BMI, weight, and height were coded as continuous variables. A Bonferroni-corrected P-value ≤ 5.0 × 10⁻³ (based on ∼10 independent tests) was used to determine statistical significance in the analysis of each risk factor and MSP levels. We also examined 26 other validated genetic risk variants for prostate cancer in association with MSP levels (12, 13, 19, 24-27). Coefficients of determination (r²) were estimated in multivariate models to assess the variance of MSP levels explained by rs10993994 genotype and other covariates.

The mean ages at the time of blood draw were well matched between racial/ethnic groups; African Americans were slightly younger on average (mean age, 60.2 y) and Japanese Americans slightly older (mean age, 61.5 y; Table 1). There were significant differences in mean BMI among racial/ethnic groups (P = 1.9 × 10⁻³), with Japanese Americans having the lowest mean BMI (25.0 kg/m²) and Latinos the highest (26.5 kg/m²). There were also significant differences in both weight and height among the groups. We observed significant differences in geometric mean MSP levels adjusted for age, BMI, laboratory batch, and free PSA across racial/ethnic groups (P = 2.0 × 10⁻⁴). On average, European Americans had the highest levels (20.4 ng/mL), followed by Latinos (19.7 ng/mL) and African Americans (19.0 ng/mL), with the lowest levels observed for Japanese Americans (14.8 ng/mL). Untransformed mean MSP levels were highest in European Americans (mean, 24.2 ng/mL; SD, 15.9) and lowest in Japanese Americans (mean, 19.2; SD, 20.4) (Supplementary Table S1).

The risk allele at rs10993994 (T allele) was common in all populations and ranged in frequency from 0.36 in Latinos to 0.55 in African Americans. The variant, rs10993994, was strongly and highly statistically associated with MSP levels in each racial/ethnic group (all global P values < 4 × 10⁻⁹; Fig. 1). Compared with men with the CC genotype, men with the CT genotype had a percent change in geometric mean PSA levels between −24.3% (African Americans) and −41.5% (Latinos; all P < 0.05), and men with the TT genotype had changes ranging from −62.3% in African Americans to −74.2% in Japanese Americans (all P < 10⁻⁸; Table 2). We observed little evidence of heterogeneity in the association of genotype and MSP levels between racial/ethnic groups (P = 0.37; Table 2). The associations were similar when adjusting for global European ancestry in the admixed African American and Latino populations (Supplementary Table S2). Differences in the frequency of rs10993994 genotype could not explain the ethnic differences in MSP levels (global P = 3.5 × 10⁻⁶; Table 1). After adjustment for genotype, MSP levels were highest in African Americans (19.3 ng/mL) and lowest in Japanese Americans (14.1 ng/mL; Table 1). We did not observe evidence that age, BMI, or PSA modified the association between MSMB genotype and geometric mean MSP levels.

We found no significant association between age and MSP levels (P = 0.10); however, the youngest 5-year age group (50-54 y) was noted as having lower least squares geometric mean levels than the older groups (55-69 y; Table 3). We observed inverse associations of MSP levels with BMI (P = 7.4 × 10⁻³) and weight (P = 3.6 × 10⁻³), and a suggestive inverse association with height (P = 0.072). Least squares geometric mean MSP levels by quintiles of these factors are shown in Table 3. Family history of prostate cancer was positively associated with MSP levels in African Americans (P = 0.023) but not in the pooled population (P = 0.34). We observed modest yet statistically significant positive correlations between MSP levels and free (P = 3.2 × 10⁻⁹) and total (P = 4.2 × 10⁻⁵) PSA, with Pearson correlation coefficients of 0.15 (0.07-0.21 among racial/ethnic groups) and 0.13 (0.08-0.19 among racial/ethnic groups), respectively. Least squares geometric mean MSP levels by quintiles of the PSA measures are shown in Table 3.

We found no strong associations between suspected risk factors for prostate cancer and MSP levels (Supplementary Table S3). Of the dietary variables, we observed a −0.44% decrease in MSP levels per 1 unit (100 mcg/kcal/d) increase in lycopene intake, which was only nominally statistically significant (P = 0.047; see Methods). In testing 26 validated risk variants (12, 13, 19, 24-27) for association with MSP levels, we observed a nominally significant inverse association with only 2 variants, rs721048 on chromosome 2p15 [EHBP1 gene locus; −11.5% change in MSP (ng/mL) per allele; P = 0.013] and rs7931342 on chromosome 11q13 (7.2% change in MSP per allele; P = 0.041).

In unadjusted models, rs10993994 genotype explained between 30% (African Americans) and 50% (Japanese Americans) of the variation in log MSP levels. Together with age, BMI, free PSA, and laboratory batch, between 39% (African Americans) and 61% (Japanese Americans) of the variation in log MSP levels could be explained. In ethnic-pooled analysis, rs10993994 explained 38% of the variance in log MSP levels, whereas 48% of the variance could be explained when including age, BMI, free PSA, ethnicity, and laboratory batch.

In this study, we found that rs10993994 genotype is an important and highly statistically significant predictor of circulating MSP levels. The association of genotype and MSP levels was observed in all racial/ethnic groups, which supports fine-mapping studies pinpointing rs10993994 as the most strongly associated prostate cancer risk variant in the region (15, 16). We also found MSP levels to vary significantly among racial/ethnic groups and after conditioning on genotype, African Americans were observed to have the highest levels and Japanese the lowest.

The variant is located in a putative CREB transcription factor binding site, and the C to T change at this position has been shown to influence CREB binding, with preferential binding observed with the low-risk C allele. In cell lines, the C allele has also been associated with increased MSMB expression (16). A recent study in a Chinese Han population found significantly lower circulating serum MSP levels in men with prostate cancer and the CT or TT genotype (16.32 μg/L) compared with men with the CC genotype (19.33 μg/L; P = 0.022; ref. 17). They also noted a similar but nonsignificant association in 30 unaffected men. In Japanese Americans we observed geometric mean levels of 6.7, 16.4, and 25.8 (ng/mL) in men with the TT, CT, and CC genotypes, respectively. Whereas our findings are similar in direction, it is difficult to compare the absolute MSP values between the studies because the MSP values reported by our new sandwich-type immunoassay are markedly higher (close to 3-fold) compared with the competitive assay design used in prior studies (21), although the MSP values reported by the two different assay designs have excellent correlation (r² > 0.90). Our observation among unaffected men from multiple populations provides further support for the hypothesis that variant rs10993994 is biologically functional and that the risk for prostate cancer may be conferred by altering the expression of MSP.

Numerous studies have investigated the genetic basis underlying interindividual variation in circulating levels of biomarkers with relevance to prostate cancer, including PSA, sex hormone-binding globulin, testosterone, 3α-androstanediol-glucoronide, and 17β-estradiol (28, 29). The genetic variants identified to date in association with these biomarkers, however, explain only a small fraction of the variation in circulating levels in any population. In contrast, the variant at the MSMB locus alone may account for 30% (African Americans) to 50% (Japanese Americans) of the variation in levels in any one population. This finding supports those of Xu et al. (30) in a population of 18-to-21-year-old Swedish men (article cosubmitted). Whereas rs10993994 does explain a large portion of the variance in MSP levels, a portion of the remaining variance may be explained by day-to-day interindividual variation, environmental risk factors, as well as genetic variation, both local to the MSMB region and genome-wide. Extensive sequencing and fine-mapping efforts of the MSMB region (31, 32) have identified additional local variation that will need to be examined for association with MSP levels in future experiments.

In previous analyses of the Multiethnic Cohort data (33), we did not replicate reports (34, 35) that high lycopene intake or blood measurements may be protective for prostate cancer, and we note here that the suggestive weak inverse association between MSP and lycopene intake is not consistent with such protection. However, the association with lycopene seen here (P = 0.047) should be interpreted cautiously because this is not significant after adjusting for multiple tests (Bonferroni-corrected α = 5.0 × 10⁻³). The geometric mean MSP levels were lower in men with more risk alleles for rs721048, which lies in an intron of the EHBP1 gene on chromosome 2, and MSP levels were higher in men with more risk alleles of rs7931342, located in a gene desert on chromosome 11. These associations also do not survive correction for multiple comparisons and will require replication.

In summary, we observed a strong association between rs10993994 genotype and plasma levels of MSP in a multiethnic sample of men without prostate cancer. The association was robust and statistically significant in all four racial/ethnic groups. The variant explained a large proportion of the variability in plasma MSP levels in all groups. Examining MSP in a prospective study will be necessary to determine whether the prostate cancer risk association with rs10993994 is mediated through influence on MSP levels, as well as the population risk associated with MSP levels.

Hans Lilja: ownership interest, Arctic Partners.

We thank Gun-Britt Eriksson and Mona Hassan Al-Battat for expert assistance with immunoassays.

Grant Support: The Multiethnic Cohort was supported by National Cancer Institute grants CA63464 and CA54281. This research was supported in part by National Cancer Institute grant P30 CA008748 (Memorial Sloan Kettering Cancer Center); National Cancer Institute Specialized Programs of Research Excellence grant P50-CA92629; Swedish Cancer Society grant 08-0345; Swedish Research Council Medicine grant K2009-54X-20095-04-3]; the Sidney Kimmel Center for Prostate and Urologic Cancers; David H. Koch through the Prostate Cancer Foundation, Academy of Finland project 206690; and Fundación Federico SA.

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