Moderate Expression of Prostate-Specific Membrane Antigen, a Tissue Differentiation Antigen and Folate Hydrolase, Facilitates Prostate Carcinogenesis

Prostate cancer is the most commonly diagnosed cancer and second leading cause of cancer death in American men (1). When compared with all other cancers, the incidence of prostate carcinoma increases most rapidly with age (2). Successful management and elimination of recurrent and metastatic prostate cancer continues to present a major clinical challenge (3).

PSMA, a type II membrane glycoprotein, is expressed in the human prostate and at lower levels in the brain, spine, peripheral nervous system, kidney, small intestine, and the salivary gland (4). PSMA is expressed in the luminal epithelial cells of normal and hyperplastic prostatic tissues, Prostatic Intraepithelial Neoplasia (PIN), and in primary and metastatic prostate cancers (5). Its expression in adenocarcinoma correlates with stage of disease and Gleason score (6, 7). PSMA expression is also higher in cancer cells from hormone-refractory patients (7, 8). Increased PSMA expression is an independent marker of recurrence (9, 10). Interestingly, endothelial cells of the neovasculature of almost all solid tumors express PSMA but not cells in the neovasculature associated with normal tissues (11–14).

PSMA has glutamate carboxypeptidase II activity that cleaves α-linked glutamate from n-acetylaspartyl glutamate (NAALADase activity; ref. 15) and γ-linked glutamates from polyglutamated folates sequentially (folate hydrolase activity; refs. 16, 17). Prostate cancer tissues have significantly elevated levels of PSMA enzymatic activity compared with normal prostate and BPH tissues (18). Increase in both expression and enzymatic activity of PSMA in aggressive prostate tumors imply that PSMA provides a selective advantage for cells expressing it, thereby contributing to the development and progression of prostate cancer.

Transgenic mice have been generated that express human PSMA in their ventral, lateral, and dorsal prostate lobes under the control of the human prostate-specific antigen promoter and enhancer (19). The level of PSMA expressed in the prostates of the transgenic mice is comparable with that in normal human prostate tissues. The level of folate hydrolase activity in the prostate of the transgenic mice is similar to the endogenous level seen in the murine brain and is significantly greater than in the prostates of nontransgenic mice. Characterization of PSMA-transgenic mice found that their prostates started to develop PIN-like lesions as early as age 21 weeks, with the majority (above 60%) developing PIN-like lesions by age 9 months. Prostates of the wild-type siblings did not exhibit PIN lesions (19). The presence of PIN-like lesions in PSMA-transgenic mice suggests that PSMA plays a role in the control of cell division or transformation, and as such the PSMA-transgenic mouse could be a suitable model for examining potential causes of prostate carcinogenesis. However, the length of time for PIN-like lesions to arise in the majority of animals is too long for the model to be cost effective, and thus far, the model has not progressed from PIN lesions to cancer.

The aim of this study was to determine if PSMA is involved in prostate carcinogenesis and progression. We used the prostatic reconstruction model (20) comprising of rat urogenital mesenchyme (rUGM) and prostate tissues from PSMA-transgenic mice or their wild-type siblings grafted beneath the renal capsule of immunodeficient mouse hosts and given pharmacologic doses of hormones to achieve our objective. Similar models have been used to show that the absence of Rb gene may predispose prostatic epithelial cells to carcinogenesis (21); and to drive PIN lesions in the prostates of Nkx3.1 knockout mice to a progressively severe histopathologic phenotype (22). Using this model, we were able to elicit carcinogenesis in a timely manner and significantly advance the understanding of the role that PSMA plays in prostate cancer.

We also explored the mechanism by which PSMA plays a role in prostate cancer. Previously, we showed that PSMA provides a growth advantage for prostate cancer cells in medium containing low and physiologic folate levels (23). Ghosh and colleagues (24) found that PSMA-expressing PC-3 cells have reduced invasive capability in RPMI 1640. However, this medium contains ∼2.3 μmol/L of folic acid, which is ∼100 times the level found at physiologic levels. Therefore, we examined the invasive ability of PC-3 cells that do not express PSMA and those that express PSMA in physiologically relevant folate levels.

PSMA-transgenic mouse prostate tissues. Animals were housed in the University of Pittsburgh at the Department of Urology. All animal experiments were approved by and followed the guidelines set out by Institutional Animal Care and Use Committee. PSMA-transgenic mice and their wild-type siblings were kindly provided by Dr. Warren Heston (The Cleveland Clinic, Cleveland, OH). Mice (age matched, 27- to 33-wk-old) were killed, and prostates were harvested and microdissected to separate the prostate lobes.

Tissue separation, recombination, and grafting. Rat urogential sinuses were removed from 18-d embryonic fetuses of pregnant Sprague-Dawley rats (Taconic) and separated into epithelial and mesenchyme components as previously described (20). Prostate lobes from PSMA-transgenic mice and wild-type siblings were cut into small (0.2–0.5 mm) pieces and combined with 250,000 rUGM cells in 40 μL neutralized type I rat tail collagen gels as previously described (25). The gels of PSMA-transgenic or wild-type prostate tissue recombinants were incubated overnight in RPMI 1640 (Cambrex Corp.) containing 5% fetal bovine serum (FBS; Atlanta Biological) and 10⁻⁸M testosterone (Sigma). Tissue recombinants were grafted beneath the renal capsule of adult male nude (Nu/Nu) CD1 mice (Charles River Laboratories) as described previously (20). UGM alone was grafted to determine contamination from urogenital epithelium.

Host nude mice carrying PSMA-transgenic or wild-type prostate tissue recombinants were treated hormonally at the time of grafting by implanting s.c. a Silastic capsule [1.54-mm inside diameter and 3.18-mm outside diameter (Dow-Corning Co.)] tightly packed with 25 mg of testosterone propionate (T) and 2.5 mg of 17β-estradiol benzoate (E2; Sigma). Eight weeks postgrafting, host nude mice were killed and each of tissue recombinant was carefully dissected from the host kidney, weighed, and cut into small pieces (0.5–2 mm). Tissue pieces were either paraffin embedded or recombined with fresh rUGM cells in collagen gels and then regrafted for a further 8 wk under the renal capsule of male nude mice. After a total of 16 wk under the renal capsule, the tissue recombinants were recombined with fresh rUGM to again grown under the renal capsule of male nude mouse hosts for a further 8 wk. Animal were treated hormonally with fresh T&E2 upon each sequential serial grafting.

Histopathology and hoechst 33258 dye staining. Tissue sections were stained with H&E for histopathology and Hoechst dye 33258 (Calbiochem) to confirm that the epithelium was of mouse origin and the stroma of rat origin (26). H&E and CK14-stained slides were examined blindly by a pathologist to determine the incidence of normal, hyperplasia, PIN or carcinoma. The most severe histologic phenotype in each tissue recombinant was recorded. PIN-like lesions were scored blindly as 1+, 2+, or 3+ in increasing severity based on their glandular, architectural, and cytologic changes by a pathologist (Dr. A. Parwani).

Immunohistochemical staining and proliferation index. Tissue sections were stained using the Vector M.O.M Peroxidase kit (Vector Laboratories). Nonimmune mouse IgG alone served as the control. Primary antibodies include anti-PSMA (PEQ226.5; Hybritech, Inc.), anti-Cytokeratin 18 (CK18; clone ks18.04; Research Diagnostics, Inc.), anti-CK14 (clone LL001; Abcam), anti–E-cadherin (clone 36; BD Transduction), anti-smooth muscle α-actin (clone 1A4; Sigma), and anti–Ki-67 (clone B56; Research Diagnostics, Inc.). Sections were developed with Vector NovaRED Substrate kit (Vector Laboratories). Proliferation index was compiled by manually counting total epithelial nuclei and epithelial nuclei showing positive immunoreactivity against Ki-67 in microscopic fields. Six to 15 samples were examined using two fields per sample. All photomicrographs were taken using Zeiss Axioplan 2 imaging microscope with an Axiocam and an Axio-Vision software.

Anchorage-independent growth. Tissue recombinants (20 mg) that had been grafted under the kidney capsule for 24 wk were digested with 108 U collagenase for at least 4 h at 37°C and seeded in 6-well plates in a double layer agar culture system. The underlay contained culture medium with a final agar concentration of 0.6% (Difco Laboratories) and the overlay contained culture medium with a final agar concentration of 0.3%. The dishes were incubated at 37°C in a humidified atmosphere, and colonies of >30 cells were counted after 21 d. Photomicrographs were captured on a Nikon T1-SM microscope.

Matrigel invasion assay. PC-3 cells transfected with pLNCX2-PSMA vector or pLNCX2 control vector were maintained in RPMI 1640 [2.3 μmol/L folic acid, called “high folate” (HF)] supplemented with 10% FBS (1.8–2 ng/mL), l-glutamine (2 mmol/L), and penicillin/streptomycin. Folate-free RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin was called “low folate” (1 nmol/L folic acid) medium. Cell invasion assay was performed using the BD BioCoat Matrigel Invasion Chambers (BD Biosciences) as per manufacturer's instructions. Folate-free RPMI 1640 supplemented with 10% FBS, ∼25 nmol/L HF medium, l-glutamine, and penicillin and streptomycin was called “physiologic folate” (25 nmol/L folic acid) medium (27). Approximately 2.5 × 10⁴ cells were seeded onto each Matrigel-coated or control (uncoated; 8 μm pore) inserts. The inserts contained either high folate or low folate medium without FBS, whereas the wells were filled with either high folate or low folate medium containing 10% FBS so that the FBS act as the chemoattractant. The chambers were incubated for 22 h at 37°C in a humidified atmosphere of 5% CO₂. After incubation, the noninvading cells were removed from the upper surface of the membrane by scrubbing with a cotton-tipped swab. The cells that invaded into the lower compartment were stained with 0.5% Crystal Violet/20% methanol and counted. All experiments were done in triplicates. This assay was performed on two different clones of vector-only or PSMA vector transfected PC-3 cells. Data are expressed as the percent invasion through the Matrigel Matrix membrane relative to the migration through the control membrane.

Statistical methods. Statistical analysis on wet weight and proliferation index of the tissue recombinants were done using the Student's t test. Wilcoxon Mann-Whitney test was used to analyze incidence of PIN 1+, 2+, and 3+ in wild-type and PSMA-transgenic tissue recombinants. The Fisher's exact test was used to analyze the incidence of PIN and foci with features of adenocarcinoma in wild-type and PSMA-transgenic tissue recombinants. The Jonckheere-Terpstra test was used to determine whether there is histopathologic progression with serial grafting of the tissue recombinants.

To test if PSMA is involved in prostate carcinogenesis, we grafted tissue recombinants consisting of PSMA-positive or nontransgenic (wild-type) prostate tissues with rUGM under the kidney capsule of host mice also implanted with Silastic tubes containing T&E2. It has previously been shown that implantation of T&E2-filled silastic capsules into nude mice significantly increases their plasma testosterone levels at 1 month and estradiol levels at 2 and 4 months postimplantation (28). Tissue recombinants were harvested after 8, 16, and 24 weeks under the kidney capsule. At each time point, the rUGM control grafts weighed significantly less (P < 0.05) than recombinants that included PSMA-transgenic or wild-type prostate tissues (Table 1). There was no significant difference in the wet weights between PSMA-transgenic and wild-type tissue recombinants; however, the wet weights of both declined with passage (Table 1). The wild-type recombinants grafted for 8 weeks had a larger mean wet weight (103.2 ± 5.2 mg) than recombinants grown for 16 weeks (70.8 ± 11.4 mg), although this did not reach statistical significance (P > 0.05). By 24 weeks, the mean wet weight (57.3 ± 8.3 mg) had decreased even further, resulting in a statistically significant difference in size between wild-type tissue recombinants at week 8 and 24 (P < 0.01). Similarly, for PSMA-transgenic tissue recombinants, the mean wet weight at 8 weeks (90.2 ± 7.4 mg) was significantly larger than the mean wet weight at 16 weeks (59.6 ± 6.5 mg; P < 0.01) and at 24 weeks (62.8 ± 8.7 mg; P < 0.05). The decline in wet weights with serial grafting may be due to the limited proliferative capacity of the majority of cells in the tissue recombinants. At no time did the tissue recombinants grow beyond the immediate area of the renal capsule and invade the host kidney.

Both wild-type and PSMA-transgenic tissue recombinants formed large masses of well-differentiated prostatic glands surrounded by dense sheets of stromal cells when grown for 8, 16, and 24 weeks under the renal capsule (Fig. 1A,, left). As determined by Hoechst 33258 nuclear staining, the epithelial cells were of mouse origin, whereas the stroma was mostly of rat origin (Fig. 1A,, right). Their genotypes of wild-type and PSMA-transgenic tissue recombinants were confirmed by staining with anti-human PSMA antibody (Fig. 1B,, top left and top right). Prostatic glands were lined by glandular epithelial cells that expressed CK18 (Fig. 1B,, bottom left) and surrounded by stromal cells that expressed smooth muscle α-actin (Fig. 1B , bottom right).

We determined the proliferation index of the recombinant tissue by dividing the number of epithelial cells with Ki-67–stained nuclei by the total number of epithelial cells (Fig. 1C,, left). As shown in Fig. 1C (right), the mean proliferation index of PSMA-transgenic tissue recombinants was greater than that of wild-type tissue recombinants at week 8, 16, and 24, although this did not reach statistical significance.

Analysis of H&E and CK14 staining of PSMA-transgenic tissue recombinants revealed the presence of small atypical glands with features of adenocarcinoma at 16 and 24 weeks (see arrows in Fig. 2). These recombinants also displayed hyperplastic ducts and PIN-like lesions at 8, 16, and 24 weeks (Fig. 2). Ducts with nuclear stratification and/or crowding and rare CK14-expressing basal cells were classified as PIN lesions, whereas adenocarcinoma were defined as small atypical glands with prominent nuclei and nucleoli as well as absence of basal cells as evidenced by negative CK14-staining. PIN-like lesions and some of the small atypical glands expressed E-cadherin along adjacent epithelial cell membranes (Fig. 2). However, other small atypical glands displayed abnormal and variable (weak membrane or cytoplasmic) E-cadherin staining (see arrows in Fig. 2).

In contrast, at no point did we observe adenocarcinoma in the wild-type prostate tissue recombinants, although we did observe normal, hyperplastic, and focal PIN-like glandular structures (Fig. 2). Normal glands were lined by a single layer of columnar epithelial cells with a continuous layer of CK14-expressing basal epithelial cells. Hyperplastic ducts were lined by multilayered luminal epithelium with a patchy layer of basal cells. Some glands displayed features typical of human PIN, including cribriform, tufted, or papillary architecture of epithelial cells with atypical and hyperchromatic nuclei, enlarged nucleoli (one or more), and mitotic bodies. CK14 staining showed that the PIN-like glands had a patchy basal cell layer (Fig. 2). E-cadherin was expressed along adjacent epithelial cell membranes (Fig. 2).

We next compared the histopathologic features of both types of recombinants at each time point. Although a recombinant may display glands with all histologic types (normal, hyperplasia, PIN, and small atypical glandular foci with features of adenocarcinoma), only the most advanced histologic phenotype found in each of the tissue recombinant was noted. As shown in Table 2, the majority of wild-type tissue recombinants displayed minor PIN-like lesions (PIN 1 and 2) at 8 (78%), 16 (64%), and 24 (79%) weeks. More importantly, none (0%) of the wild-type tissue recombinants displayed glandular foci with features of adenocarcinoma. In contrast, a variable number of small atypical glandular foci with features of adenocarcinoma were present in PSMA-transgenic tissue recombinants at 8 (4%), 16 (30%), and 24 (46%) weeks. This difference is statistically significant at 16 (P = 0.046) and 24 (P = 0.012) weeks but not at 8 (P = 0.719) weeks, as determined by the Fisher's exact test. In addition, unlike wild-type tissue recombinants, PSMA-transgenic tissue recombinants displayed a statistically significant (P = 0.0014) progression from PIN lesions to foci suspicious for adenocarcinoma with serial grafting, as determined by the Jonckheere-Terpstra Test. This is illustrated by a concomitant increase in the number of tissue recombinants with small atypical glands with features of adenocarcinoma (4%, 29%, and 46%) and a decline in the number of recombinants with PIN lesions (96%, 71%, and 50%) as their worst histology over the course of the experiment.

Although both wild-type and PSMA-transgenic tissue recombinants displayed PIN-like lesions, the cytologic and pathologic atypia of PIN-like lesions in wild-type tissue recombinants were less severe than those in PSMA-transgenic tissue recombinants. PIN-like lesions of both wild-type and PSMA-transgenic prostate tissue recombinants were scored blindly by a single pathologist. The classification of PIN lesions was based on their architectural and cytologic atypia. The scale ranged from 1+ to 3+, with 3+ representing the most severe histopathology (see Supplementary Data). PIN-like lesions scored as 1+ (least severe) displayed prostatic hyperplasia with the glandular epithelial cells having uniform oval nuclei, some of which were slightly enlarged and/or exhibited weak hyperchromasia. Also, nucleoli were either absent or inconspicuous. PIN-like lesions with a score of 2+ (moderately severe) consisted of glands with moderate hyperplasia, larger cells, higher nucleus to cytoplasmic ratios, more prominent nucleoli, and more pronounced hyperchromasia. Cells with an overall papillary-type architecture displayed more crowding and stratification. Occasionally, a tufted type of architecture was also noted. PIN-like lesions with a score of 3+ (most severe) were characterized by a pronounced increase in cell size, more pleomorphism, striking hyperchromasia with enlarged nucleoli, and some cells contained more than one nucleoli—a feature more commonly seen in neoplasia. There was significant architectural disorder, including the presence of papillary tufts. At low power, these foci were very prominent due to the overall dark, hyperchromatic appearance of the nuclei, giving them an overall dark blue appearance in contrast to adjacent benign glandular structures. In addition, there was significant stratification of the nuclei, giving the appearance of multilayering.

Table 3 summarizes the incidence of 1+, 2+, and 3+ PIN-like in wild-type and PSMA-transgenic tissue recombinants. PIN-like lesions present in wild-type tissue recombinants never scored 3+ in severity. In contrast, in the PSMA-transgenic tissue recombinants, a score of 3+ was assigned to 23% (5 of 22), 25% (4 of 16), and 54% (7 of 13) of PIN-like lesions at 8, 16, and 24 weeks, respectively. The incidence of PIN 3+ (most severe) in PSMA-transgenic tissue recombinants is statistically significant from wild-type prostate tissue recombinants at 8 (P = 0.00046), 16 (P = 0.0334), and 24 (P = 0.0000021) weeks as analyzed with the Wilcoxon Mann-Whitney test statistics. In addition, serial grafting led to a statistical significant (P = 0.0336) increase in the number of PSMA-transgenic tissue recombinants with PIN 3+ (most severe) as analyzed with the Jonckheere-Terpstra Test.

A hallmark characteristic of tumor cells is their ability to grow in an anchorage-independent manner (29). As shown in Fig. 3A, cells from PSMA-transgenic tissue recombinants, which had been grafted under the kidney capsule for 24 weeks, were able to grow and form an average of ∼5 colonies of >30 cells in semisolid agar. In contrast, cells from wild-type tissue recombinants, which had been grafted under the kidney capsule for 24 weeks, were not able to form colonies of >30 cells in semisolid agar.

We explored the mechanism by which PSMA may influence carcinogenesis by testing the invasive ability of PC-3 cells that do not express PSMA and those that express PSMA in physiologically relevant folate levels. As shown in Fig. 3B, we found that the invasive ability of PC-3 cells with ectopic expression of PSMA were not statistically significant compared with PC-3 cells that do not express PSMA in standard medium, which contains high (2.3 μmol/L; percentage invasion: 5.0% ± 0.6% and 11.0% ± 3.8% versus 7.3.% ± 0.9% and 7.3% ± 4.1%) folate levels. However, PSMA-expressing PC-3 cells had increased invasive ability compared with nonexpressing PC-3 cells in medium containing low (<1 nmol/L; 51.7% ± 13.0% and 36.0% ± 8.7% versus 6.2% ± 4.9% and 2.6% ± 3.1%) and physiologic folate levels (25 nmol/L; percentage invasion: 14.6% ± 3.9% and 11.9% ± 2.5% for PSMA-expressing PC-3 cells versus 1.2% ± 0.2 and 5.8% ± 2.1% for PC-3 cells transfected with vector only; P < 0.05). Two clones of PSMA-expressing PC-3 cells and vector-transfected PC-3 cells were used to confirm the results.

PSMA-transgenic mice provide tantalizing preliminary evidence that PSMA contributes to prostate cancer development. However, PIN-like lesions in PSMA-transgenic mice did not progress to cancer. Given that carcinogenesis requires multiple insults, these lesions may not have acquired all of the underlying molecular traits necessary for malignant transformation. Previous work has shown that the tissue recombinant model provides the additional factors needed for cancer development. This model combines hormonal induction of prostate cancer (30–33) with enhanced epithelial proliferation elicited by embryonic rUGM (34–36). We used this model to more definitively evaluate the role of PSMA in prostate carcinogenesis and progression.

Prostatic hyperplasia, PIN, and foci with features of adenocarcinoma were observed in PSMA-transgenic tissue recombinants. In contrast, PIN 2+ was the most severe lesion observed in wild-type tissue recombinants, which may be induced by 17β-estradiol. Only PSMA-transgenic tissue recombinants developed PIN with the most severe architectural and cytologic atypia, PIN 3+. Serial grafting of the PSMA-transgenic, but not wild-type tissue recombinants, resulted in a statistically significant progression in the architectural and cytologic severity from PIN 2+ to PIN 3+. In addition, serial grafting led to progression of the PIN-like lesions in PSMA-transgenic tissue recombinants progressed to small atypical glands with features of adenocarcinoma.

At each time point, the PSMA-transgenic tissue recombinants developed small atypical glands that appeared similar to adenocarcinoma. Such small atypical glands were absent in wild-type prostate tissue recombinants and prostate tissues from PSMA-transgenic mice. These small atypical glands did not form large masses of back-to-back glands but existed in isolation or in clusters of two to four glands. They exhibited features of adenocarcinoma such as nuclear pleomorphism, prominent and often multiple nucleoli, loss of CK14-expressing basal cells, and aberrant E-cadherin staining. Loss or abnormal E-cadherin expression has been associated with metastasis and poor prognosis (37). The fact that we were able to grow these small atypical cells in semisolid medium (anchorage-independent growth) provides further evidence of their tumorigenicity (38). Similar to Wang and colleagues (21), we were able to transplant the small atypical glands. Moreover, their number increased with serial grafting, suggesting neoplastic progression with successive passage.

In this model, the level of human PSMA expressed in PSMA-transgenic prostates is comparable with that found in normal human prostate tissues. In addition, no other genetic manipulations were made that would not be present in normal human prostate tissue samples before cancer development. Therefore, formation of PIN-like lesions and foci with features of adenocarcinoma in PSMA-transgenic prostate recombinants suggests that PSMA, thought to be a cell differentiation marker and not an oncogene or cell cycle regulator, may be an initiator of prostate carcinogenesis.

Previously, Ghosh and colleagues (24) found that ectopic expression of PSMA in PC-3 cells decreased their invasive capability, whereas knockdown of PSMA expression increased the invasiveness of LNCaP cells. They also found that the enzymatic activity of PSMA is associated with the effect of PSMA on invasion. However, their study was conducted in RPMI 1640, which contains ∼100 times the physiologic folate level. At this concentration, the enzymatic site of PSMA, a folate hydrolase, may be competitively occupied by folic acid, thereby, masking the affects of PSMA. Therefore, we decided to look at the effects of ectopic PSMA expression on the invasive ability of PC-3 cells in RPMI 1640 containing low (<1 nmol/L), physiologic (25 nmol/L), and high (2.3 μmol/L) folate levels. In contrast to Ghosh and colleagues (24), we counted the number of invading cells 22 hours instead of 48 hours after seeding. We found that PC-3 cells expressing PSMA have greater invasive ability than nonexpressing cells in medium containing low and physiologic folate levels but not in medium containing high folate levels. Increased invasive ability was most prominent in RPMI 1640 containing low (<1 nmol/L) levels of folic acid where prostate cancer cells expressing PSMA had ∼10-fold greater percentage invasion than prostate cancer cells transfected with vector only. Based on the data presented here and from our previous study (23), PSMA expression may increase the invasiveness of prostate cancer cells and provide cells with a growth advantage in the low folate tumor microenvironment. The observation that PSMA expression increases invasion, at least under physiologic folate conditions is not without precedent. Conway and colleagues (39) found that angiogenesis is impaired in PSMA-null animals due to reduced invasion of endothelial cells through extracellular matrix.

In conclusion, we have shown that PSMA, expressed at a level comparable with that of normal human prostate tissue and not mutated in any way, can initiate the development of prostate cancer using a hormone-induced tissue recombination model. PSMA may facilitate carcinogenesis by enhancing the proliferative and invasive capability of prostate cancer cells. This invasive capability can be blocked by folic acid. These findings show a novel mechanism that may contribute to current knowledge about the role of folate in cancer prevention and could potentially lead to the use of PSMA inhibitors as novel chemotherapeutic agents for prostate cancer. Our laboratory is currently using a compound that can inhibit the enzymatic activity of PSMA to investigate if it contributes to the initiation of prostate carcinogenesis.

No potential conflicts of interest were disclosed.

Grant support: Department of Defense Prostate Cancer Research Program under the award number W81XWH-05-1-0015. Views, opinions, and endorsements by the authors do not reflect those of the US Army or the Department of Defense. This work was also supported by a grant from the AFUD/AUAER Research Scholar Program and the Reston Ambassador's Golf Classic for Prostate Cancer Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Simon Hayward for his technical advice and our laboratory technician Diana Sisca Harya.