MSMB Variation and Prostate Cancer Risk: Clues Towards a Possible Fungal Etiology

Abstract

BACKGROUND

With recent advances in high-throughput sequencing technologies, many prostate cancer risk loci have been identified, including rs10993994, a single nucleotide polymorphism (SNP) located near the MSMB gene. Variant allele (T) carriers of this SNP produce less prostate secretory protein 94 (PSP94), the protein product of MSMB, and have an increased risk of prostate cancer (approximately 25% per T allele), suggesting that PSP94 plays a protective role in prostate carcinogenesis, although the mechanisms for such protection are unclear.

METHODS

We reviewed the literature on possible mechanisms for PSP94 protection for prostate cancer.

RESULTS

One possible mechanism is tumor suppression, as PSP94 has been observed to inhibit cell or tumor growth in in vitro and in vivo models. Another novel mechanism, which we propose in this review article, is that PSP94 may protect against prostate cancer by preventing or limiting an intracellular fungal infection in the prostate. This mechanism is based on the recent discovery of PSP94’s fungicidal activity in low-calcium environments (such as the cytosol of epithelial cells), and accumulating evidence suggesting a role for inflammation in prostate carcinogenesis. We provide further details of our proposed mechanism in this review article.

CONCLUSIONS

To explore this mechanism, future studies should consider screening prostate specimens for fungi using the rapidly expanding number of molecular techniques capable of identifying infectious agents from the entire tree of life.

INTRODUCTION

Recent advances in high-throughput sequencing technologies have allowed for the identification of at least 77 genetic loci associated with prostate cancer risk in genome-wide association and replication studies [1]. These recent genetic discoveries offer the potential for risk prediction, targeted prevention and screening, biomarker development, and insight into prostate cancer etiology. Loci identified to date include, most notably, single nucleotide polymorphisms (SNPs) in the 8q24 and 17q12 chromosomal regions, and those related to the HNF1B and MSMB genes [2]. While SNPs in the 8q24 region have received much attention, our review article focuses on genetic variation related to the MSMB gene and proposes a novel hypothesis as to how this variation may inform a fungal etiology of prostate cancer.

MSMB AND PROSTATE CANCER RISK

Two genome-wide association and several replication studies have observed associations between SNP rs10993994, a SNP located 57 base pairs upstream of the first exon of MSMB in its proximal promoter region [3], and prostate cancer risk [4–8]. In the largest replication study, CC homozygotes had the lowest risk of prostate cancer, heterozygotes had an intermediate risk (odds ratio [OR] = 1.21; 95% CI = 1.11–1.33), and TT homozygotes had the highest risk of prostate cancer (OR = 1.57; 95% CI = 1.42–1.74) [4]; similar associations were observed in smaller studies and among men of varying races [7–14]. Results from fine-mapping studies further indicate that associations between rs10993994 carrier status and prostate cancer risk were stronger and more significant than those for other local tag SNPs, which, together with observed promoter activity, suggest that rs10993994 is one of the main SNPs responsible for observed associations between MSMB-related variation and prostate cancer risk [3,8]. Finally, although rs10993994 carrier status was also associated with levels of mRNA for NCOA4 (a gene located within 16 kb telomeric of rs10993994) in one study of benign prostate tissue [15], no association was observed for NCOA4 protein levels in the only other study to examine this possible relation [16], suggesting that rs10993994 genetic associations are likely specific to MSMB.

While the association between rs10993994 carrier status and prostate cancer risk is certainly strong, reproducible, and likely specific for MSMB, one concern for all genetic association studies is the possibility of prostate-specific antigen (PSA) detection bias. This bias occurs when the variant under investigation is associated with prostate cancer detection through its association with elevated PSA levels and the likelihood of prostate biopsy independent of the presence of prostate cancer. Although rs10993994 carrier status is associated with PSA levels [6,12,16–18], we believe that PSA detection bias is unlikely to explain associations between rs10993994 status and prostate cancer risk because similar associations were observed for both cancers more and less likely to be detected purely by PSA (e.g., low-grade, low-stage, and non-palpable disease, and cancers detected after the introduction of PSA screening vs. high-grade, high-stage, palpable, and pre-PSA era disease) in most previous studies [4,5,7–9,11,13], and because no association was observed with the likelihood of a negative prostate biopsy in one further study [18]. African-American men, who have the highest risk of prostate cancer diagnosis and mortality [19], are also more likely to carry the high-risk T allele (60% in African- American controls [7] vs. 40% in their Caucasian counterparts [5,6]), further arguing against a major influence of detection bias. Thus together, these findings support a causal role for MSMB-related variation in prostate carcinogenesis.

PROSTATE SECRETORY PROTEIN 94

MSMB encodes prostate secretory protein 94 (PSP94), a 94-amino acid, 10.7 kDa member of the immunoglobulin binding factor family and one of the three main proteins secreted by prostate epithelial cells into seminal plasma [20]. Over the years, this protein has had several different names and hypothesized functions. It was first identified in semen in the early 1980s and named by two independent groups. One group reported that PSP94 suppressed the secretion of follicle stimulating hormone (FSH), suggesting a role for PSP94 as an inhibin, and named it β-inhibin [21]. Shortly thereafter, however, FSH suppression could not be reproduced [22,23], and the name prostate secretory protein 94 was adopted instead [24]. A second group reported that PSP94 coated the surface of spermatozoa, suggesting a role in reproduction, and named it β-microseminoprotein (β-MSP, MSP, or MSMB) [25]. This potential role for PSP94 is still being explored [26].

In addition to its possible involvement in reproduction, recent associations between MSMB-related variation and prostate cancer risk clearly suggest a role for PSP94 in prostate cancer development. Based on levels of MSMB expression among men of varying rs10993994 genotype, this role appears to be protective. Low-risk rs10993994 C allele carriers have the highest levels of promoter activity [3,8] and prostate tissue expression [15,16,27], and the highest concentrations of PSP94 in their blood [13,17], urine [27], and seminal plasma [8,17,27], suggesting that high PSP94 expression may be protective for prostate cancer. Although the protective mechanisms conferred by the rs10993994 C allele are unknown, two previously hypothesized biological functions of PSP94 could explain this effect: tumor suppression [28] and pathogen defense [29]. These two hypothesized functions are reviewed here in detail; a brief review of all hypothesized functions for PSP94 was published by Lazure et al. [30].

Tumor Suppression

In 1992, Lokeshwar et al. investigated the effects of seminal plasma on prostate cancer cells and reported strong inhibition of human PC3 and DU145 cells, as well as rat DT cells, which they attributed to TGF-β₁ (for all three cell types [31]) and PSP94 (for DT cells only [32]). This was the first report, to our knowledge, of PSP94’s tumor suppression properties. Thereafter, many studies have continued to explore its tumor suppression properties and those of its derived peptides using in vitro and rodent models [15,28,32–39]. Considering findings from in vitro studies, generally variable results have been observed across models and peptides. Three studies examined the effects of human PSP94 on PC3 cells, two of which reported no growth inhibition [28,37], while a third reported strong inhibition [33]. Another study examined two additional human prostate cancer cell lines, and reported moderate growth inhibition of LNCaP cells and strong inhibition of WPE1-NB26 cells [37]. Finally, one further study suppressed MSMB expression in immortalized prostate epithelial cells and found that such suppression promoted anchorage-independent growth [15]. All additional studies explored the influence of PSP94 or derived peptides on rat prostate cancer cells. One study reported strong growth inhibition by human PSP94 and peptides derived from PSP94 amino acids 31–45, but not amino acids 7–21 and 76–94, on the MLL rat prostate cancer cell line [34,35]; another observed no inhibition by human PSP94 on the MLL cell line [28]; and a third study observed strong inhibition by amino acids 85–94 on rat DT prostate cancer cells [32]. Finally, one further study tested the influence of rat PSP94 on the PAIII rat cancer cell line and reported strong growth inhibition [36].

In contrast to in vitro findings, those from in vivo rodent models have tended to be more consistent. All six in vivo studies reported tumor suppression by subcutaneously injected human PSP94 and derived compounds in rats and mice [28,32–35,38], although only two of these studies were sufficiently similar to allow for a comparison of the minimum dose required for tumor suppression. In these two studies, the minimum dose varied from 25 [28] to 1,000 ng/kg/day [34] for tumor suppression of the MLL rat cancer cell line in male Copenhagen rats, making it difficult to comment on the minimum required dose.

Finally, in addition to findings from in vitro and rodent models, changes in PSP94 expression in the prostate with the development of malignancy may also inform its tumor suppression properties. Compared to normal prostate tissue, lower levels of MSMB mRNA and PSP94 expression have been observed consistently in localized prostate cancer lesions, although no further differences were observed by Gleason score in most previous studies [16,40–45]. These findings suggest that high PSP94 expression may influence prostate cancer development, but not prostate cancer cell differentiation, consistent with observed null associations with grade in most genetic studies [4,7,8]. Possible mechanisms that have been proposed to explain all of these findings include suppression of FSH [28,34,46], inhibition of DNA synthesis [32], failed DNA repair [47], inhibition of angiogenesis [39], and increased apoptosis of cancer cells by binding to CRISP-3 [48] or laminin receptors [49].

Beyond tumor development, further observations suggest that PSP94 may also play a role in inhibiting prostate cancer recurrence or metastasis. This role may be relevant to genetic findings for rs10993994 if properties related to prostate cancer recurrence/metastasis overlap with those related to prostate cancer development. Human PSP94 and a peptide derived from amino acids 31–45 reduced microvessel density and experimental skeletal metastases, and inhibited matrix metalloproteinase (MMP-9) mediated metastasis in in vitro and rodent models [34,35,39,50]. Lower expression of MSMB mRNA was also observed in prostate cancer metastases than in primary prostate tumors in three human tissue-based studies [51–53], and lower levels of prostatic PSP94 expression were associated with a greater likelihood of prostate cancer recurrence/metastasis in some studies [40,43], while others reported the opposite association [42,45]. These findings suggest that, in addition to suppressing tumor development, PSP94 may also contribute to inhibition of prostate cancer recurrence and metastasis.

Pathogen Defense

Another possible and novel role for PSP94 is pathogen defense, which has only recently been explored. In their study of the anti-microbial properties of semen, Hagerwall et al. [29] demonstrated potent fungicidal activity against the fungus Candida albicans in post-coital seminal plasma. This activity was mediated by PSP94 amino acids 66–76 and was limited to post-coital seminal plasma; PSP94 was not fungicidal in normal seminal plasma, which the authors attributed to selective inhibition by calcium ions at a neutral pH but not at the acidic pH of the post-coital milieu. The authors further demonstrated that porcine PSP94, which shares 51% amino acid sequence identity with human PSP94, was also fungicidal, suggesting that this may be a retained and fundamental property of PSP94. Finally, Hagerwall et al. [29] tested the toxicity of PSP94 against common bacterial uropathogens and human cells, and found it to lack both bactericidal and cytotoxic activity, which they attributed, at least in part, to its observed selective affinity for fungal plasma membranes. In liposome experiments, they found that PSP94 disrupted ergosterol-rich liposomes, which mimic the plasma membranes of most fungi and some protists, but not cholesterol-rich liposomes, which mimic mammalian plasma membranes.

Although Hagerwall et al.’s findings clearly demonstrate that PSP94 is fungicidal in the acidic, calcium ion binding-inhibiting environment of post-coital seminal plasma, whether or not it is also fungicidal in other naturally occurring low-calcium environments is largely unknown. Within the prostate, the only low-calcium ion environment is the cytosol—where calcium ions are all but absent. [54] Unfortunately, few studies have examined the intracellular localization of PSP94 to inform the possibility of cytosolic localization. In their immunoelectron microscopic study of prostate tissue from older men with benign prostatic hyperplasia (BPH) or prostate cancer, Ito et al. [55] observed PSP94 protein staining primarily in the secretory granules, but not the cytosol, of prostate epithelial cells. Similar granular staining was also observed by Doctor et al. [56] in tissue from men with BPH. However, when these investigators examined tissue from young men without prostate disease, they observed diffuse rather than granular PSP94 protein staining throughout the cytoplasm, providing support for cytosolic localization of PSP94 in the non-diseased prostate. This finding is particularly intriguing given accumulating evidence for an inflammatory, and thus possibly an infectious, contribution to prostate cancer [57].

INFLAMMATION AND PROSTATE CANCER

Similar to cancers with known inflammatory or infectious etiologies, inflammation is extremely common in the prostate [58–60]. Of relevance to prostate cancer causation, intraprostatic inflammation is also present in young men before the onset of prostate diseases [59], and is common in the peripheral zone [61], the zone at highest risk of prostate cancer development. Within this zone, inflammation tends to be observed near areas of proliferative atrophy (termed “proliferative inflammatory atrophy” lesions), which have been proposed as signatures of the “field effect” of prostate carcinogenesis (i.e., a field of cells with genomic alterations believed to predispose to prostate cancer), because a subset of these lesions share somatic DNA alterations with prostate cancer precursor lesions (high-grade prostatic intraepithelial neoplasia, HGPIN) and prostate adenocarcinomas, and because they have been observed at times in close proximity or transitioning into HGPIN and small adenocarcinomas [57]. This hypothesis is supported by findings from animal model studies, in which inflammation induced by various means has been shown to lead to epithelial hyperproliferation and atrophy, and occasionally dysplasia/PIN [57,62,63]. It is also supported by findings from a recent observational study, in which a positive association was observed between intraprostatic inflammation and high-grade prostate cancer among men without indication for biopsy—thus where the positive association between PSA and inflammation should not have influenced findings (Gurel et al. [64]). Weaker or null associations were observed for low-grade prostate cancer in this same study [64] and in another smaller study [65]. Finally, positive associations were observed between tumor-associated intraprostatic inflammation (sometimes accompanied by focal atrophy) and subsequent prostate cancer recurrence or death in most [66–70], but not all [71,72], studies of prostate cancer patients. Thus together, these findings support a role for intraprostatic inflammation in prostate carcinogenesis.

Despite this evidence, responsible causes of asymptomatic intraprostatic inflammation have not been identified, although several possibilities have been proposed. These include urine reflux, which might induce either chemical or physical trauma; trapped spermatozoa; dietary factors, such as heterocyclic amines; estrogens; infectious agents, particularly those that ascend the urethra; or a combination of several of these factors. A break in tolerance to prostate antigens triggered by any of these factors has also been proposed [57]. However, irrespective of the type of proposed inflammatory cause(s)—infectious or otherwise— it must be very common to explain the high prevalence of asymptomatic intraprostatic inflammation [58–60] and prostate cancer precursor lesions [73].

INFECTIONS AND PROSTATE CANCER

Over the past few decades, many epidemiologic studies have investigated infections in relation to prostate cancer, focused primarily on sexually transmitted infections (STIs). Findings from several of these studies have been pooled with combined results suggesting a positive association between ever having had an STI and prostate cancer (OR = 1.44–1.48 [74,75]). However, as many of these studies were case–control in design with retrospective, self-reported assessment of STI history, the possibility of recall bias has been raised (i.e., prostate cancer cases may have been more likely to respond to STI questions truthfully than controls). This concern is supported by recent null cohort study findings for several of these same self-reported STIs [76]. On the other hand, accumulating positive findings for other STIs that tend to be asymptomatic and are thus better assessed by serology than by self-report, such as Trichomonas vaginalis and mycoplasma infections [76,77], provide further sup- port for an infectious contribution to prostate cancer risk. These STIs may also be more likely to establish persistent prostate infections than those assessed by self-report because of their frequent lack of symptoms and thus lack of treatment [76]. Recent epidemiologic and/or animal model-based findings for other infectious agents not typically considered to be sexually transmitted, such as Escherichia coli and Propionibacterium acnes, are also intriguing and warrant further study [78]. However, while each of these infectious leads is promising, none is yet conclusive. It is also possible that no single infectious agent is by itself responsible for prostate cancer risk, but that many may have the potential to contribute to prostate carcinogenesis, depending on their propensity to elicit prostatic inflammation and establish long-term prostate infections.

Given recently observed associations between MSMB variation and prostate cancer, as well as the recent discovery of fungicidal activity associated with PSP94 in low-calcium environments such as the cytosol of epithelial cells, we propose that intracellular fungal infections may also be good candidate infections for prostate carcinogenesis. An intracellular infectious etiology is also consistent with the finding of a greater ratio of cytotoxic/suppressor (CD8⁺) T cells to helper/inducer (CD4⁺) T cells in the intraepithelial compartment of the prostate than in the stroma [79–81], which Bostwick et al. [80] have proposed may represent the first line of defense against foreign luminal agents. As cytotoxic T cells are known to aid in clearing intracellular pathogens, such pathogens may be promising candidate causes of intraprostatic inflammation and prostate cancer. However, while many studies have examined viral and intracellular bacterial infections [77], very few studies— only one, to our knowledge [82]—have investigated fungal infections in relation to prostate cancer. This paucity of mycotic research is likely due to the low documented occurrence of symptomatic fungal prostate infections. For instance, although Candida species, Aspergillus species, Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis are possible causes of clinical prostatitis, the fraction of prostatitis cases caused by such infections is very low [83]. These species are also readily culturable; therefore, if they were common asymptomatic pathogens, they would likely have already been identified in at least one large previous study of prostate specimen culture [84].

While findings from this previous culture study make the possibility of a culturable fungal etiology unlikely, the possibility of a fastidious fungal candidate remains plausible. For instance, microsporidia (a phylum of fastidious intracellular fungi) are known to infect healthy individuals asymptomatically [85], some species have been detected in prostate specimens [86] and urine [87], and some are suspected of a sexual route of transmission [85,86,88]. However, no studies, to our knowledge, have investigated microsporidia species in relation to prostate cancer to date, although DNA primers are available for several of these species [89]. Therefore, these microsporidia species could be explored in relation to prostate cancer in future studies.

Beyond microsporidia, it is also possible that an as-yet-undiscovered, fastidious fungal species may be involved. While not specific to fungi, as early as 12 years ago, Strickler and Goedert [90] proposed a “yet unrecognized” sexually transmissible infectious etiology for prostate cancer. This hypothesis was based on the similarity between the low estimates of association for various STIs with prostate cancer and the low estimates of association for herpes simplex virus and other STIs with cervical cancer, which was eventually attributed to their frequent co-acquisition with human papillomaviruses, the actual causal agents of cervical cancer. While this hypothesis led to many viral and bacterial screens of prostate specimens [77], no studies, to our knowledge, screened for a broad spectrum of fungi. Such screens are possible, as demonstrated recently by Ghannoum et al. [91] who used molecular techniques to identify fungi in the oral cavity of healthy individuals; these authors found 85 fungal genera of which 11 were non-culturable. These same molecular techniques could be applied to prostate specimens. However, even the consensus primers used by Ghannoum et al. cannot detect all fungal phyla (for instance, these primers do not detect microsporidia or Pneumocystis jiroveci). Therefore, newly developed techniques that can identify infectious agents from the entire tree of life (viruses, bacteria, protists, and fungi) should also be considered [92,93].

CONCLUSION

In summary, we propose a second mechanism to explain the consistent association between variation in MSMB, the gene that encodes PSP94, and prostate cancer risk [2]—in addition to PSP94’s purported tumor suppression properties. Specifically, we propose that PSP94 may protect against prostate cancer by preventing or limiting an intracellular fungal (or possibly protistan) infection in the prostate, based on the recent discovery of PSP94’s fungicidal activity in low-calcium environments, such as the cytosol of epithelial cells [29], and accumulating evidence suggesting a role for inflammation in prostate carcinogenesis [57,63]. While we have yet to test for the presence of such infectious agents in prostate specimens, some support for this idea comes from recent epidemiologic findings from the King County prostate cancer case–control study. This study observed a positive association between lifetime number of sexual partners, a marker of cumulative STI exposure, and prostate cancer among high-risk rs10993994 T allele carriers, but no association among C allele homozygotes, which produce the greatest amount of PSP94 [94]. Future studies should consider screening prostate specimens for fungi and protists, particularly fastidious ones, using the rapidly expanding number of molecular techniques capable of identifying infectious agents from the entire tree of life. These types of investigations are critical because the discovery of an infectious etiology of prostate cancer could have tremendous influence on rates of this common malignancy; such a discovery would offer the potential for primary prevention strategies using existing or newly developed anti-microbial therapies, such as pharmaceutical drugs targeting ergosterol.