Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jun;152(6):2150-63.
doi: 10.1210/en.2010-1377. Epub 2011 Mar 22.

Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells

Wen-Yang Hu  1 , Guang-Bin Shi, Hung-Ming Lam, Dan-Ping Hu, Shuk-Mei Ho, Ikenna C Madueke, Andre Kajdacsy-Balla, Gail S Prins
Affiliations

Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells

Wen-Yang Hu et al. Endocrinology. 2011 Jun.

Abstract

The present study sought to determine whether estrogens with testosterone support are sufficient to transform the normal human prostate epithelium and promote progression to invasive adenocarcinoma using a novel chimeric prostate model. Adult prostate stem/early progenitor cells were isolated from normal human prostates through prostasphere formation in three-dimensional culture. The stem/early progenitor cell status and clonality of prostasphere cells was confirmed by immunocytochemistry and Hoechst staining. Normal prostate progenitor cells were found to express estrogen receptor α, estrogen receptor β, and G protein-coupled receptor 30 mRNA and protein and were responsive to 1 nm estradiol-17β with increased numbers and prostasphere size, implicating them as direct estrogen targets. Recombinants of human prostate progenitor cells with rat urogenital sinus mesenchyme formed chimeric prostate tissue in vivo under the renal capsule of nude mice. Cytodifferentiation of human prostate progenitor cells in chimeric tissues was confirmed by immunohistochemistry using epithelial cell markers (p63, cytokeratin 8/18, and androgen receptor), whereas human origin and functional differentiation were confirmed by expression of human nuclear antigen and prostate-specific antigen, respectively. Once mature tissues formed, the hosts were exposed to elevated testosterone and estradiol-17β for 1-4 months, and prostate pathology was longitudinally monitored. Induction of prostate cancer in the human stem/progenitor cell-generated prostatic tissue was observed over time, progressing from normal histology to epithelial hyperplasia, prostate intraepithelial neoplasia, and prostate cancer with local renal invasion. These findings provide the first direct evidence that human prostate progenitor cells are estrogen targets and that estradiol in an androgen-supported milieu is a carcinogen for human prostate epithelium.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Isolation and growth of prostaspheres from primary cultures of normal human PrEC. A, Normal human PrEC grown in 2D culture were used as a source for stem/progenitor cells. A small fraction (0.2–1.0%) of PrEC was differentially selected and formed prostaspheres in a 3D matrigel culture system. At the early stage of prostasphere formation, single prostate progenitor cells divided and formed two- (B), four- (C), and eight-cell (D) structures. E, At d-3 of culture, early sphere formation was observed. F, Typical d-4 prostasphere 25–40 μm in diameter. G, Prostaspheres continued to grow, reaching approximately 60–90 μm in diameter by d-7. H, Day-7 prostasphere after 12-h bromodeoxyuridine incorporation (pink) revealed proliferation rates of 30–50% in the progenitor cells. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). I–Q, To document clonal origin of prostaspheres from single cells, equal amounts of Hoechst-labeled and -unlabeled PrEC cells were mixed and placed in 3D culture. I and J, At culture d-2, a dividing progenitor cell at two-cell stage (I) transferred Hoechst stain to the daughter cell as seen using the blue fluorescent channel (J). K–L, Culture d-4 prostasphere with a brightfield (K) or blue fluorescent channel (L) shows all progenitor cells in the spheroid positive for Hoechst blue staining. M and N, Another prostasphere in the mixed culture at d-4 (M) was entirely negative under the blue fluorescent channel (N). O–Q, Day-4 prostaspheres from mixed 3D cultures were transferred to 2D culture for overnight attachment and outgrowth. Blue fluorescent channel viewing shows Hoechst-labeled nuclei (O), whereas red fluorescent channel viewing shows propidium iodide nuclear counterstain (P). When merged (Q), all cells in a single prostasphere are shown to be Hoechst positive (pink). Scale bar, 50 μm.
Fig. 2.
Fig. 2.
A, Immunofluorescent labeling of d-7 prostaspheres with Nanog, TROP2, CD49f, SSEA4, and ABCG2 confirms their stem/early progenitor cell characteristics. The majority of prostasphere cells were positive for membrane-associated TROP2, CD49f, ABCG2, SSEA4 [with blue 4′,6-diamidino-2-phenylindole (DAPI) counterstain], and nuclear Nanog. Confocal images are shown for TROP2, CD49f, and ABCG2, whereas fluorescent imaging of whole-mounted prostaspheres was employed for Nanog and SSEA4. In the latter, excess signal in the prostasphere center is a result of the dense cell compaction in the spheroid. Scale bar, 50 μm. B, Photomicrographs showing Hoechst exclusion by d-7 prostasphere cells and 2D PrEC in the absence or presence of 50 μm verapamil. Note that the exclusion of 0.5 μg/ml Hoechst dye by prostasphere cells (upper left) was blocked by verapamil which caused the retention of Hoechst dye (upper right), indicating stem/progenitor cell nature of the cells. In contrast, no significant Hoechst dye exclusion was found in the 2D culture PrEC cells (lower left), and there was no effect of verapamil on their dye retention (lower right). Scale bar, 50 μm.
Fig. 3.
Fig. 3.
Steroid receptor expression in d-7 prostaspheres and growth response of progenitor cells to E2. A, Steroid receptor mRNA levels in d-7 prostaspheres, LNCaP, and PC3 cell lines as determined by quantitative real-time PCR. Data are normalized to LNCaP levels (set as 1) after normalization of each sample to GAPDH. Although the prostate progenitor cells did not express AR, there was robust expression of ERα, ERβ1, GPR30, and PR. ERβ expression was measured using primers specific for ERβ1 as well as primers that amplify all ERβ isoforms (ERβall); n = 3 separate experiments. B, Immunofluorescent labeling of d-7 prostasphere cells for ERα, ERβ, or GPR30. ERα and ERβ primarily localized to the nucleus, whereas GPR30 localized to the cell membrane and, to a lesser degree, cytoplasm. The inset shows fluorescence with IgG substituted for primary antibody. Scale bar, 50 μm. C, Prostaspheres were grown for 7 d in a 3D matrigel culture in the absence or presence of 1 nm E2, and prostasphere numbers and sizes (40–80 μm, >80 μm diameter) were measured. Both the number of prostaspheres that formed as well as their size at d-7 were markedly increased in response to estrogen exposure. *, P < 0.05 vs. control; n = 6 separate experiments.
Fig. 4.
Fig. 4.
In vitro differentiation of normal prostaspheres with extended culture. A, By d-10 of culture, the cells located in the prostasphere center began to differentiate, forming a double-layered structure of 100–150 μm in diameter. B, Immunofluorescent labeling of d-10 prostaspheres with p63 (green) and 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue) shows that the basal cells (aqua-green) are located in the outer layer, whereas the more centrally located cells are p63 negative. The green signal in the compacted prostasphere center was nonspecific and appeared when IgG was substituted for primary antibody (inset). C, Immunofluorescent labeling of d-10 prostaspheres with CK8 (green), NKX3.1 (red-purple), and DAPI nuclear stain (blue). The positively stained cells are located toward the prostasphere center and represent the differentiated cells. D–F, Prostaspheres cultured through d-30 began to grow laterally, forming ductal-like structures (D and E) with lumen formation (F). Scale bar, 50 μm (A–F). G, Steroid receptor and PSA expression levels in d-7 and d-30 prostaspheres (PS) and in parental PrEC grown in 2D culture as measured by real-time RT-PCR. Data were normalized to GAPDH in each sample and expressed relative to d-7 prostasphere levels set as 1. Day-30 prostasphere cells and the primary epithelial cell cultures from human prostate expressed AR, higher levels of all ER and PR, as well as PSA when compared with the undifferentiated d-7 prostasphere cells.
Fig. 5.
Fig. 5.
Characterization of chimeric prostate tissue from normal human prostate progenitor cells. A, Increasing numbers (100–10,000) of prostate progenitor cells mixed with a constant amount of UGM produced grafts of increasing size. B, H&E staining of a 1-month graft shows normal glandular structure with prostatic histology. C, Immunofluorescent labeling with antibodies against p63 (green-aqua) and CK8/18 (red) with 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei confirms differentiation of cells into prostate basal and luminal epithelial cells. D, Nuclear immunostaining of AR (green) further confirms prostatic cytodifferentiation of the epithelium. E, The human origin of the epithelium in chimeric grafts was demonstrated by immunolabeling with human-specific antinuclear antigen (red stain; pink when merged with blue nuclear DAPI) and DAPI-labeled nuclei (blue) at 1 month. Note that stromal cells are negative for human nuclear antigen. F, Human origin and functional differentiation of the epithelium were further confirmed by immunostaining for PSA (red) in the luminal cells with DAPI-labeled nuclei. Scale bar, 50 μm.
Fig. 6.
Fig. 6.
Graft biopsy after 1 and 2 months of T+E2 exposure revealed SQM and epithelial hyperplasia progressing to HG PIN over time within the same chimeric prostate graft. A, Open biopsy through ligature permitted removal of a portion of the renal graft, which was then returned to the abdominal cavity for continued growth and progression. B, The same graft was removed after 2 months of hormone treatment revealing the continued growth of the chimeric tissue. C, At 1 month of T+E2 exposure, extensive epithelial hyperplasia with infolding of ducts was observed throughout the chimeric graft. D, SQM was frequently observed after 1 month and beyond of T+E2 treatment. E, Histologic examination of the graft shown in B revealed areas of HG PIN with piling and overlapping epithelial cells, nuclear enlargement, hyperchromasia, and prominence of nucleoli. Scale bar, 50 μm.
Fig. 7.
Fig. 7.
Prostate cancer in chimeric prostate renal grafts induced by T+E2 treatment for 2–4 months. A, H&E section of graft exposed to T+E2 for 2 months reveals neoplastic epithelium with enlarged nuclei and prominent nucleoli and their local invasion into the underlying stromal region (cells highlighted with arrowheads). B, CK8/18 immunostained acini shown in A confirms the local invasion of neoplastic cells into the stroma. C and D, Within 2 months of elevated T+E2 exposure, full malignancy was induced in another chimeric renal graft as evidenced by irregular small to medium size glandular structures, abortive glandular lumen, back-to-back lumens, infiltrative glands, and loss of the basement membrane and basal cell layer. Multiple lesions of this type were observed within the graft. E and F, Adjacent sections of a chimeric prostate graft after 4 months of T+E2 treatment with H&E stain (E) and immunofluorescent labeling with CK8/18 (red) and CK14 (green) (F). Staining showed heterogeneous glandular structures with mixture of normal glands, PIN lesions, and carcinoma as well as invasion into kidney. G, Immunostaining with antibody specific to human nuclear antigen (pink nuclei, arrowheads) identifies the human origin of infiltrating epithelial cells (CK8/18+, red) within the stroma. H, Immunolabeling for PSA (red) confirms the human identity of the cancerous ducts and infiltrating cells (arrows) in the grafted tissue. Scale bar, 50 μm.
See this image and copyright information in PMC

References

    1. Prins GS. 1997. Developmental estrogenization of the prostate gland. In: Naz RK. ed. Prostate: basic and clinical aspects. Chap 10 Boca Raton, FL:CRC Press; 247–265
    1. Prins GS, Birch L, Tang WY, Ho SM. 2007. Developmental estrogen exposures predispose to prostate carcinogenesis with aging. Reprod Toxicol 23:374–382 - PMC - PubMed
    1. Prins GS, Korach KS. 2008. The role of estrogens and estrogen receptors in normal prostate growth and disease. Steroids 73:233–244 - PMC - PubMed
    1. Ellem SJ, Risbridger GP. 2007. Treating prostate cancer: a rationale for targeting local oestrogens. Nat Rev 7:621–627 - PubMed
    1. Leung YK, Lam HM, Wu S, Song D, Levin L, Cheng L, Wu CL, Ho SM. 2010. Estrogen receptor β2 and β5 are associated with poor prognosis in prostate cancer, and promote cancer cell migration and invasion. Endocr-Relat Cancer 17:675–689 - PMC - PubMed

Publication types

MeSH terms