Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder

Adult bladder epithelium (BLE) is induced to differentiate into glandular epithelium after association with urogenital sinus mesenchyme (UGM) and subsequent in vivo growth in syngeneic male hosts. Alteration of epithelial cytodifferentiation is associated with the expression of prostate-specific antigens, histochemical and steroid metabolic activities. These observations suggest that the inductive influence of the UGM has reprogrammed both the morphological and functional characteristics of the urothelium. In this report, differences regarding the mechanisms and effects of androgenic stimulation of prostate and bladder are exploited to determine the extent to which UGM plus BLE recombinants express a prostatelike, androgen-dependent phenotype. Results from cytosolic and autoradiographic binding studies suggest that androgen binding is induced in UGM plus BLE recombinants and that this activity is accounted for by the induced urothelial cells. In UGM plus BLE recombinants, androgen-induced [3H]thymidine or [35S]-methionine uptake analyzed by two-dimensional gel electrophoresis was qualitatively and quantitatively similar to that of prostate as opposed to bladder. These studies indicate that expression within BLE of prostatic phenotype is associated with a loss of urothelial characteristics and that androgen sensitivity is presumably a function of the inductive activities of the stroma.     

Urothelial transdifferentiation to prostate epithelia is mediated by paracrine TGF-β signaling

The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF-β signaling directed the prostatic induction. UGM from TGF-β type II receptor stromal conditional knockout mice (Tgfbr2^fspKO) or control mice (Tgfbr2^{floxE2/floxE2}) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2^{floxE2/floxE2} UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2^fspKO UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2^fspKO UGM compared with Tgfbr2^{floxE2/floxE2} UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF-β activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF-β signaling to mediate epithelial Wnt activity.     

Gene expression profiling identifies a unique androgen-mediated inflammatory/immune signature and a PTEN (phosphatase and tensin homolog deleted on chromosome 10)-mediated apoptotic response specific to the rat ventral prostate

Understanding androgen regulation of gene expression is critical for deciphering mechanisms responsible for the transition from androgen-responsive (AR) to androgen-independent (AI) prostate cancer (PCa). To identify genes differentially regulated by androgens in each prostate lobe, the rat castration model was used. Microarray analysis was performed to compare dorsolateral (DLP) and ventral prostate (VP) samples from sham-castrated, castrated, and testosterone-replenished castrated rats. Our data demonstrate that, after castration, the VP and the DLP differed in the number of genes with altered expression (1496 in VP vs. 256 in DLP) and the nature of pathways modulated. Gene signatures related to apoptosis and immune response specific to the ventral prostate were identified. Microarray and RT-PCR analyses demonstrated the androgen repression of IGF binding protein-3 and -5, CCAAT-enhancer binding protein-delta, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) genes, previously implicated in apoptosis. We show that PTEN protein was increased only in the luminal epithelial cells of the VP, suggesting that it may be a key mediator of VP apoptosis in the absence of androgens. The castration-induced immune/inflammatory gene cluster observed specifically in the VP included IL-15 and IL-18. Immunostaining of the VP, but not the DLP, showed an influx of T cells, macrophages, and mast cells, suggesting that these cells may be the source of the immune signature genes. Interestingly, IL-18 was localized mainly to the basal epithelial cells and the infiltrating macrophages in the regressing VP, whereas IL-15 was induced in the luminal epithelium. The VP castration model exhibits immune cell infiltration and loss of PTEN that is often observed in progressive PCa, thereby making this model useful for further delineation of androgen-regulated gene expression with relevance to PCa.     

Suppressed prostate epithelial development with impaired branching morphogenesis in mice lacking stromal fibromuscular androgen receptor

Using the cre-loxP system, we generated a new mouse model [double stromal androgen receptor knockout (dARKO)] with selectively deleted androgen receptor (AR) in both stromal fibroblasts and smooth muscle cells, and found the size of the anterior prostate (AP) lobes was significantly reduced as compared with those from wild-type littermate controls. The reduction in prostate size of the dARKO mouse was accompanied by impaired branching morphogenesis and partial loss of the infolding glandular structure. Further dissection found decreased proliferation and increased apoptosis of the prostate epithelium in the dARKO mouse AP. These phenotype changes were further confirmed with newly established immortalized prostate stromal cells (PrSC) from wild-type and dARKO mice. Mechanistically, IGF-1, placental growth factor, and secreted phosphoprotein-1 controlled by stromal AR were differentially expressed in PrSC-wt and PrSC-ARKO. Moreover, the conditioned media (CM) from PrSC-wt promoted prostate epithelium growth significantly as compared with CM from PrSC-dARKO. Finally, adding IGF-1/placental growth factor recombinant proteins into PrSC-dARKO CM was able to partially rescue epithelium growth. Together, our data concluded that stromal fibromuscular AR could modulate epithelium growth and maintain cellular homeostasis through identified growth factors.     

Sex steroid receptor expression and localization in benign prostatic hyperplasia varies with tissue compartment

Androgens and estrogens, acting via their respective receptors, are important in benign prostatic hyperplasia (BPH). The goals of this study were to quantitatively characterize the tissue distribution and staining intensity of androgen receptor (AR) and estrogen receptor-alpha (ERα), and assess cells expressing both AR and ERα, in human BPH compared to normal prostate. A tissue microarray composed of normal prostate and BPH tissue was used and multiplexed immunohistochemistry was performed to detect AR and ERα. We used a multispectral imaging platform for automated scanning, tissue and cell segmentation and marker quantification. BPH specimens had an increased number of epithelial and stromal cells and increased percentage of epithelium. In both stroma and epithelium, the mean nuclear area was decreased in BPH relative to normal prostate. AR expression and staining intensity in epithelial and stromal cells was significantly increased in BPH compared to normal prostate. ERα expression was increased in BPH epithelium. However, stromal ERα expression and staining intensity was decreased in BPH compared to normal prostate. Double positive (AR and ERα) epithelial cells were more prevalent in BPH, and fewer double negative (AR and ERα) stromal and epithelial negative cells were observed in BPH. These data underscore the importance of tissue layer localization and expression of steroid hormone receptors in the prostate. Understanding the tissue-specific hormone action of androgens and estrogens will lead to a better understanding of mechanisms of pathogenesis in the prostate and may lead to better treatment for BPH.     

Regenerated luminal epithelial cells are derived from preexisting luminal epithelial cells in adult mouse prostate

Determining the source of regenerated luminal epithelial cells in the adult prostate during androgen deprivation and replacement will provide insights into the origin of prostate cancer cells and their fate during androgen deprivation therapy. Prostate stem cells in the epithelial layer have been suggested to give rise to luminal epithelium. However, the extent of stem cell participation to prostate regrowth is not clear. In this report, using prostate-specific antigen-CreER(T2)-based genetic lineage marking/tracing in mice, preexisting luminal epithelial cells were shown to be a source of regenerated luminal epithelial cells in the adult prostate. Prostatic luminal epithelial cells could survive androgen deprivation and were capable of proliferating upon androgen replacement. Prostate cancer cells, typically exhibiting a luminal epithelial phenotype, may retain this intrinsic capability to survive and regenerate in response to changes in androgen signaling, providing part of the mechanism for the ultimate failure of androgen deprivation therapy in prostate cancer.     

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Background  

Androgens and androgen receptors (AR) regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA). This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells.     

Immunolocalization of tenascin-C, alpha9 integrin subunit, and alphavbeta6 integrin during wound healing in human oral mucosa.

Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal-epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-C(L)) and the putative TN-C-binding alpha9 and alphavbeta6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for alpha9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-C(L) were localized in the matrix between and underneath alpha9-expressing epithelial cells. In parallel with gradual downregulation of alpha9 integrin immunoreactivity in 7-day and older wounds, the expression of alphavbeta6 integrin was temporarily induced. Integrin alphavbeta6 co-localized in the same area as TN-C and TN-C(L) immunoreactivity at the cell-cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-C(L) were abundantly localized in the granulation tissue. The findings show that TN-C(L) is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of alpha9 integrin in migrating epithelial cells and of alphavbeta6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.     

Anterior prostate epithelial AR inactivation modifies estrogen receptor expression and increases estrogen sensitivity

Androgens influence prostate growth and development, so androgen withdrawal can control progression of prostate diseases. Although estrogen treatment was originally used to induce androgen withdrawal, more recently direct estrogen effects on the prostate have been recognized, but the nature of androgen-estrogen interactions within the prostate remain poorly understood. To characterize androgen effects on estrogen sensitivity in the mouse prostate, we contrasted models of castration-induced androgen withdrawal in the prostate stromal and epithelial compartments with a prostate epithelial androgen receptor (AR) knockout (PEARKO) mouse model of selective epithelial AR inactivation. Castration markedly increased prostate epithelial estrogen receptor (ER)α immunoreactivity compared with very low ERα expression in intact males. Similarly, strong basal and luminal ERα expression was detected in PEARKO prostate of intact males, suggesting that epithelial AR activity regulated epithelial ERα expression. ERβ was strongly expressed in intact, castrated, and PEARKO prostate. However, strong clusters of epithelial ERβ positivity coincided with epithelial stratification in PEARKO prostate. In vivo estrogen sensitivity was increased in PEARKO males, with greater estradiol-induced prostate growth and epithelial proliferation leading to squamous metaplasia, featuring markedly increased epithelial proliferation, thickening, and keratinization compared with littermate controls. Our results suggest that ERα expression in the prostate epithelial cells is regulated by local, epithelia-specific, androgen-dependent mechanisms, and this imbalance in the AR- and ER-mediated signaling sensitizes the mature prostate to exogenous estrogens.     

Experimental endocrine therapies promote epithelial cytodifferentiation and ciliogenesis in the gerbil female prostate

The incidence of ciliated cells in the prostate gland of the female gerbil (Meriones unguiculatus) is uncommon and apparently becomes more frequent during androgen (testosterone cypionate) and anti-estrogen (letrozole) endocrine therapies. To evaluate the effects of such drug therapies on the induction of ciliogenesis in the glandular epithelium of female prostate glands, adult female gerbils aged 90 days were treated for 14 days with testosterone and letrozole after which their prostate glands were removed for histological, ultrastructural, and serological analyses. The cytodifferentiation of the ciliated phenotype in the alveolar epithelium became more frequent after both the testosterone and the letrozole treatments. The ciliogenesis phenomenon of the epithelial cells in the prostate gland of female gerbils thus appears to be induced by variations in the increase of androgen levels.     

Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control

Metastasis is the primary cause of death in prostate cancer (PCa) patients. Effective therapeutic intervention in metastatic PCa is undermined by our poor understanding of its molecular aetiology. Defining the mechanisms underlying PCa metastasis may lead to insights into how to decrease morbidity and mortality in this disease. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a potent precursor of advanced glycation end products (AGEs). Hydroimidazolone (MG‐H1) and argpyrimidine (AP) are AGEs originating from MG‐mediated post‐translational modification of proteins at arginine residues. AP is involved in the control of epithelial to mesenchymal transition (EMT), a crucial determinant of cancer metastasis and invasion, whose regulation mechanisms in malignant cells are still emerging. Here, we uncover a novel mechanism linking Glo1 to the maintenance of the metastatic phenotype of PCa cells by controlling EMT by engaging the tumour suppressor miR‐101, MG‐H1‐AP and TGF‐β1/Smad signalling. Moreover, circulating levels of Glo1, miR‐101, MG‐H1‐AP and TGF‐β1 in patients with metastatic compared with non‐metastatic PCa support our in vitro results, demonstrating their clinical relevance. We suggest that Glo1, together with miR‐101, might be potential therapeutic targets for metastatic PCa, possibly by metformin administration.     

Transforming growth factor beta 1 and androgen receptors in prostate neoplasia

OBJECTIVE: To investigate the interplay between transforming growth factor (TGF) beta 1, androgen receptors and stromal-epithelial interactions in benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma areas of prostate neoplasia. STUDY DESIGN: In this immunohistochemical study we investigated staining patterns and then determined the correlation between TGF-beta 1 expression and androgen receptor status in the epithelium and stroma of 60 paraffin-embedded tissues from radical prostatectomies. RESULTS: Staining patterns differed in the epithelium and stroma of tumor and peritumor prostatic tissue. TGF-beta 1 immunostaining (H-scores) in the epithelium and stroma increased significantly from BPH to PIN and from BPH to prostate carcinoma in the epithelium (P < .05), whereas androgen receptor (AR) immunoreactivity significantly (P < .05) increased from BPH to PIN to prostatic carcinoma in epithelium and stroma. TGF-beta 1 did not correlate with histologic grade of differentiation, whereas AR proteins were more strongly expressed in Gleason score 5 and 6 than score 7 tumors (P < .05). Nonlinear regression showed a significant correlation (P < .01) between TGF-beta 1 and AR expression only in the stromal compartment of PIN. CONCLUSION: These findings argue in favor of an interaction between TGF-beta 1 and AR in the early stages of prostate carcinogenesis and suggest that TGF-beta 1 plays a central role in stromal-epithelial interactions during the early stages of malignant transformation.     

Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer

Drug resistance is a major limitation to the successful treatment of advanced prostate cancer (PCa). Patients who have metastatic, castration‐resistant PCa (mCRPC) are treated with chemotherapeutics. However, these standard therapy modalities culminate in the development of resistance. We established paclitaxel resistance in a classic, androgen‐insensitive mCRPC cell line (DU145) and, using a suite of molecular and biophysical methods, characterized the structural and functional changes in vitro and in vivo that are associated with the development of drug resistance. After acquiring paclitaxel‐resistance, cells exhibited an abnormal nuclear morphology with extensive chromosomal content, an increase in stiffness, and faster cytoskeletal remodeling dynamics. Compared with the parental DU145, paclitaxel‐resistant (DU145‐TxR) cells became highly invasive and motile in vitro, exercised greater cell traction forces, and formed larger and rapidly growing tumors in mouse xenografts. Furthermore, DU145‐TxR cells showed a discrete loss of keratins but a distinct gain of ZEB1, Vimentin and Snail, suggesting an epithelial‐to‐mesenchymal transition. These findings demonstrate, for the first time, that paclitaxel resistance in PCa is associated with a trans‐differentiation of epithelial cell machinery that enables more aggressive and invasive phenotype and portend new strategies for developing novel biomarkers and effective treatment modalities for PCa patients. J. Cell. Biochem. 114: 1286–1293, 2013. © 2012 Wiley Periodicals, Inc.     

Localization of annexins I, II, IV and VII in whole prostate sections from radical prostatectomy patients

Annexins (ANXs) represent a family of calcium and phospholipid binding proteins that are involved in several physiological processes e.g. signal transduction, cellular differentiation and proliferation. Since they are known to be dysregulated in a variety of cancers we investigated the immunolocalization of ANXs in whole prostate sections containing benign prostatic epithelium (BPE), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) in order to evaluate their possible role during tumorigenesis. Samples were obtained from 28 patients undergoing radical prostatectomy. Gross sections of whole prostates were examined immunohistochemically for the distribution of ANX I, II, IV and VII. In BPE all ANXs were localized to the cell membranes and the cytoplasm of all gland cells. In BPH the immunoreactivity of ANX I and II was restricted to the basal cells of glands and expression pattern of ANX IV and VII was similar to BPE. In PIN only basal cells expressed ANX II. In PCa ANX II immunoreactivity was absent and weak ANX I and ANX IV immunoreactivity was restricted to the cytoplasm of tumor cells. ANX VII immunoreactivity was seen in some but not all tumor cells. Since ANX IV and VII expression did not show significant changes in PCa compared to non-neoplastic tissue and PIN an essential role during prostate tumourigenesis seems unlikely. In contrast, as progression from PIN to PCa is characterized by a reduction of ANX I and II this suggests that downregulation of these proteins could represent an important event in prostate carcinogenesis.     

The role of protein tyrosine phosphatases in prostate cancer biology

Prostate cancer (PCa) is the most frequent malignancy in the male population of Western countries. Although earlier detection and more active surveillance have improved survival, it is still a challenge how to treat advanced cases. Since androgen receptor (AR) and AR-related signaling pathways are fundamental in the growth of normal and neoplastic prostate cells, targeting androgen synthesis or AR activity constitutes the basis of the current hormonal therapies in PCa. However, resistance to these treatments develops, both by AR-dependent and -independent mechanisms. Thus, alternative therapeutic approaches should be developed to target more efficiently advanced disease. Protein tyrosine phosphatases (PTPs) are direct regulators of the protein- and residue-specific phosphotyrosine (pTyr) content of cells, and dysregulation of the cellular Tyr phosphorylation/dephosphorylation balance is a major driving event in cancer, including PCa. Here, we review the current knowledge on the role of classical PTPs in the growth, differentiation, and survival of epithelial prostate cells, and their potential as important players and therapeutic targets for modulation in PCa.     

Immunohistochemical expression and localization of somatostatin receptors in normal prostate, high grade prostatic intraepithelial neoplasia and prostate cancer and its many faces

Data on the immunohistochemical expression and localization of the five somatostatin receptors (SSTRs) have been obtained by our group in separate studies concerning the many faces of prostate cancer (PCa), its precursor high grade prostatic intraepithelial neoplasia (HGPIN) and normal epithelium (Nep). This publication highlights the key findings, with special reference to: normal prostate epithelium; untreated HGPIN and PCa, both clinically and incidentally detected; PCa with NE differentiation; HGPIN and PCa following complete androgen ablation (CAA); and hormone refractory (HR) PCa. Taken together, the data obtained in these investigations demonstrate that SSTR profiling in individual patients with HGPIN and the multifaceted PCa is feasible and is of relevance to better tailor the somatostatin analogue-based treatment.     

Androgen Regulated Genes in Human Prostate Xenografts in Mice: Relation to BPH and Prostate Cancer

Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues.     

Characterization of an in vitro model to study the possible role of polyomavirus BK in prostate cancer

Prostate cancer (PCa) is the most common male neoplasms in the Western world. Various risk factors may lead to carcinogenesis, including infectious agents such as polyomavirus BK (BKPyV), which infects the human renourinary tract, establishes latency, and encodes oncoproteins. Previous studies suggested that BKPyV plays a role in PCa pathogenesis. However, the unspecific tropism of BKPyV and the lack of in vitro models of BKPyV-infected prostate cells cast doubt on this hypothesis. The aim of the present study was to determine whether BKPyV could (a) infect normal and/or tumoral epithelial prostate cells and (b) affect their phenotype. Normal epithelial prostate RWPE-1 cells and PCa PC-3 cells were infected with BKPyV for 21 days. Cell proliferation, cytokine production, adhesion, invasion ability, and epithelial-to-mesenchymal transition (EMT) markers were analyzed. Our results show that (a) RWPE-1 and PC-3 cells are both infectable with BKPyV, but the outcome of the infection varies, (b) cell proliferation and TNF-α production were increased in BKPyV-infected RWPE-1, but not in PC-3 cells, (c) adhesion to matrigel and invasion abilities were elevated in BKPyV-infected RWPE-1 cells, and (d) loss of E-cadherin and expression of vimentin occurred in both uninfected and infected RWPE-1 cells. In conclusion, BKPyV may change some features of the normal prostate cells but is not needed for maintaining the transformed phenotype in the PCa cells The fact that RWPE-1 cells exhibit some phenotype modifications related to EMT represents a limit of this in vitro model.