Moderate expression of prostate-specific membrane antigen, a tissue differentiation antigen and folate hydrolase, facilitates prostate carcinogenesis

Abstract

Increased expression of PSMA, a differentiation antigen with folate hydrolase activity, is an independent marker of prostate cancer progression. Mice expressing moderate levels of human PSMA in their prostate develop PIN-like lesions by 9 months. The aim of this study was to determine whether PSMA is involved in prostate carcinogenesis and progression and, if so, the possible mechanism by which PSMA may exert its effects. Using prostates from PSMA-transgenic mice, we developed a tissue recombinant model that exhibits small atypical glands with features of adenocarcinoma. This was not observed in tissue recombinants that were composed of prostate tissues from the wild-type siblings. Cells from PSMA-transgenic tissue recombinants have the ability to form colonies in semisolid agar. PSMA may facilitate this phenotype by increasing the invasive ability of cells. Ectopic PSMA expression on PC-3 cells increased the invasive capacity of cells in in vitro invasion assays, which could be competed out by folic acid. These results suggest PSMA facilitates the development of prostate cancer, and the invasive ability of these cells may be modulated by folate levels. These findings show a novel mechanism that may contribute to the known role of folate in cancer prevention, and may lead to the use of PSMA inhibitors as novel chemopreventive agents for prostate cancer. Moreover, our model should prove useful for further dissecting pathways involved in prostate carcinogenesis and progression.

Figure 1

Histology and proliferation index of wild-type and PSMA-transgenic tissue recombinants. The urogenital mesenchyme microdissected from embryonic day 18 rat fetuses were combined with prostates from either wild-type or PSMA-transgenic tissues and grafted underneath the kidney capsules. A, Wild-type and PSMA-transgenic tissue recombinants harvested from a representative host mouse showing size, 8 weeks after kidney grafting (left). Hoechst 33258 staining of paraffin embedded tissue recombinants show that the epithelial cells were of mouse origin because they contained several small discrete intranuclear fluorescent bodies whereas their absence in stromal cells indicates that they are of rat origin (right). B, Immunohistochemical staining of paraffin embedded wild-type (top left) and PSMA-transgenic (top right) tissue recombinants with monoclonal anti-mouse PSMA antibody show PSMA expression only in PSMA-transgenic recombinants. Immunohistochemical staining of paraffin embedded tissue recombinants with CK-18 and smooth muscle α-actin monoclonal antibodies show CK18-expression in luminal epithelial cells (bottom left) and α-actin expression in the stromal cells surrounding glandular structures (bottom right). Photomicrographs were captured at 20× magnification. C, Immunohistochemical staining of paraffin embedded tissue recombinants with an anti-mouse Ki-67 monoclonal antibody show strong brown nuclear staining of cells undergoing proliferation. Arrows point to Ki-67-positive cells (left). The cells positive for Ki-67 were counted by monitoring at least 200 luminal epithelial cells for normal, PIN and carcinoma lesions in multiple regions of the same sample (right). There was no difference in the proliferation index of wild-type and PSMA-transgenic tissue recombinants grafted beneath the kidney capsule of intact nude mice hosts treated with T&E2 for 8, 16 and 24 weeks. Data are reported as mean ± SE (n = 7) by Student t-test. Photomicrograph was captured at 40× magnification.

Figure 2

PSMA expression promotes cancer formation in mouse prostate recombinants. Paraffin embedded tissue recombinants grafted for a total of 8, 16 and 24 weeks were stained with either CK14 or E-cadherin. A pathologist (Dr. A Parwani) graded all specimens in blinded fashion. Arrows point to small atypical glands that lack CK-14 (basal cell) expression and display aberrant E-cadherin expression. These prostate glands display features of carcinoma similar to that of human prostate adenocarcinoma. Only PSMA-transgenic tissue recombinants were observed to display these small atypical glands with features of adenocarcinoma. Wild-type tissue recombinants displayed PINlike lesions that have strong CK14- and membrane E-cadherin expressions. Photomicrographs were captured at 40× magnification.

Figure 3

PSMA expression facilitates formation of cells with the ability to grow in an anchorage-independent manner and ectopic expression of PSMA on prostate cancer cell line increases invasion in media containing physiological level of folate. A, cells isolated from PSMA-transgenic tissue recombinants that had been grafted under the kidney capsule for 24 weeks were able to form colonies of greater than 30 cells on semi-sold agar (left). In contrast, cells isolated from wild-type tissue recombinants did not form colonies on semi-solid a gar (right). Photomicrographs were captured at 40× magnification. B. PC-3 cells expressing PSMA displayed increased invasive ability than PC-3 transfected with vector only in media containing physiological (25nM) and low (< 1nM) folate levels compared to those grown in high (normal media, 2.3µM) folate media. Transwell migration assay was performed using a Boyden chamber system. Data is reported as percent invasion through the matrigel matrix and membrane relative to the migration through the control membrane; mean ±SE (n = 3) * denotes p<0.05, by Student t-test.