Interferon-gamma and its functional receptors overexpression in benign prostatic hyperplasia and prostatic carcinoma: parallelism with c-myc and p53 expression

The therapeutic potential of IFN-gamma in prostatic cancer has been documented in several reports, although no immunohistochemical studies of this factor and its receptors in the prostate have been reported. The aim of the present study was to investigate the expression of IFN-gamma and its receptor components (IFN-gamma-Ralpha and IFN-gamma-Rbeta) in normal prostate, benign prostatic hyperplasia (BPH) and prostatic cancer (PC), as well as the possible relationship between this factor and the products of the p53 gene (the wild and mutant forms) and the oncogene c-myc, by means of immunochemical techniques (Western blot, ELISA, and quantification of immunostaining in histological sections). In normal prostate, IFN-gamma and its two receptors were expressed in the basal cells of the epithelium and some stromal cells. In BPH specimens, immunostaining of basal epithelial cells was significantly increased for IFN-gamma and its a receptor, whereas stromal cell immunostaining was significantly increased for IFN-gamma and its b receptor. In addition, columnar epithelial cells immunostained for IFNbeta-Rbeta. PC specimens differed from BPH specimens in the significantly increased immunostaining of epithelial cells for IFN-gamma and its two receptors, and the immunostaining of columnar epithelial cells for IFN-gamma-Ralpha. Immunodetection of wild-p53 was weak and limited to some stromal cells in the three types of specimens. Immunostainings for both mutant-p53 and c-myc were negative in normal prostate, and positive in the epithelium and stromal cells of both BPH and PC specimens. Immunostaining intensity in PC was significantly higher than in BPH. These observations suggest that the expression of both mutant-p53 and c-myc, together with other factors, might be involved in the development of prostatic hyperplasia and neoplasia, while the increased expression of IFN-gamma and its receptors could be regarded as an attempt, although insufficient, to inhibit the uncontrolled cell proliferation.     

Gene targeting to the stroma of the prostate and bone

Abstract Stromal–epithelial interactions mediated by paracrine signaling mechanisms dictate prostate development and progression of prostate cancer. The regulatory role of androgens in both the prostate stromal and epithelial compartments set the prostate apart from many other organs and tissues with regard to gene targeting. The identification of androgen-dependent prostate epithelial promoters has allowed successful gene targeting to the prostate epithelial compartment. Currently, there are no transgenic mouse models available to specifically alter gene expression within the prostate stromal compartment. As a primary metastatic site for prostate cancer is bone, the functional dissection of the bone stromal compartment is important for understanding stromal–epithelial interactions associated with metastatic tumor growth. Use of currently available methodologies for the expression or deletion of gene expression in recent research studies has advanced our understanding of the stroma. However, the complexity of stromal heterogeneity within the prostate remains a challenge to obtaining compartment or cell-lineage-specific in vivo models necessary for furthering our understanding of prostatic developmental, benign, tumorigenic, and metastatic growth.     

Integrating Biomedical Knowledge to Model Pathways of Prostate Cancer Progression

Due to pathologic, histologic, and biologic variation within prostate cancers, profiling the genetic changes associated with disease progression has been difficult. Although initial integration of data from profiling studies had been limited by platform variation, bioinformatic tools and analytic techniques have enabled integrative analysis of profiling studies and the identification of more robust and valid profiles. The identification of key transition points in the progression of prostate cancer relies on profiling precursor lesions and “pure” cell populations. Utilizing laser-capture microdissection to isolate 101 cell populations, a more specific genetic profile of progression from benign epithelium to metastatic disease was obtained. This laser-capture profile was analyzed in the context of the Molecular Concepts Map (MCM), a compendium of over 15,000 molecular concepts including other expression profiles of prostate cancer, to obtain an integrative molecular model of progression. The conceptual connections associated with progression confirm that prostate cancer biology is largely driven by pathways related to androgen signaling and epithelial cell biology; however, further analysis of concepts associated with progression suggests stromal factors are highly associated with progression of prostate cancer. The effect of stromal signatures on the progression model suggests the impact of stromal signature downregulation may reflect both a change in the epithelia:stroma ratio within higher grade tumors and also a microenvironment influence on prostate epithelia. Analyzing complex gene expression signatures in the context of molecular concepts improves integrative models and may improve detection, prognostication, or targeted therapy.     

前列腺组织中神经生长因子(NGF)表达的研究

研究前列腺组织中神经生长因子（ＮＧＦ） 的生理学意义。采用原位杂交和免疫组化法, 检测４３ 例前列腺增生组织, ８ 例腺癌组织和８ 例正常组织中β－ＮＧＦｍ ＲＮＡ及其蛋白的表达及分布。结果显示β－ＮＧＦｍ ＲＮＡ 在正常组织及增生组织中定位于间质细胞, 偶见于上皮细胞中; 而在癌组织中, 上皮细胞和间质细胞有同样强度的β－ＮＧＦｍ ＲＮＡ染色。其蛋白在良性组织中表达主要着色在间质细胞中,上皮细胞呈弱表达,而癌组织中上皮细胞见着色明显增强（Ｐ＜ ０．０５）。ＮＧＦ的自分泌异常可见是前列腺组织由良性向恶性转变的原因之一。     

Reference spectra from squamous epithelium and connective tissue allow whole section proteomics analysis

We reasoned that micro-dissection of tumour cells for protein expression studies should be omitted since tumour-stroma interactions are an important part of the biology of solid tumours. To study such interactions in head and neck squamous cell carcinoma (HNSCC) development, we generated reference protein spectra for normal squamous epithelium and connective tissue by SELDI-TOF-MS. Calgranulins A and B, Annexin1 and Histone H4 were found to be strongly enriched in the epithelium. The alpha-defensins 1-3 and the haemoglobin subunits were identified in the connective tissue. Tumour-distant epithelia, representing early pre-malignant lesions, showed up-regulated expression of the stromal alpha-defensins, whereas the epithelial Annexin 1 was down-regulated. Thus, tumour microenvironment interactions occur very early in the carcinogenic process. These data demonstrate that omitting micro-dissection is actually beneficial for studying changes in protein expression during development and progression of solid tumours.     

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.     

An absence of stromal caveolin-1 is associated with advanced prostate cancer,metastatic disease spread and epithelial Akt activation

Here, we examined the status of stromal Cav-1 expression in patients with benign prostatic hypertrophy (BPH), primary prostate cancers (PCa), and prostate-cancer metastases (Mets). Interestingly, an absence of stromal Cav-1 directly correlated with prostate cancer disease progression. For example, virtually all BPH samples showed abundant stromal Cav-1 immunostaining. In contrast, in a subset of patients with primary prostate cancer, the stromal levels of Cav-1 were significantly decreased, and this correlated with a high Gleason score, indicative of a worse prognosis and poor clinical outcome. Remarkably, all metastatic tumors (either from lymph node or bone) were completely negative for stromal Cav-1 staining. Thus, stromal Cav-1 expression may be considered as a new biomarker of prostate cancer disease progression and metastasis. Mechanistically, stromal Cav-1 levels were inversely correlated with the epithelial expression levels of Cav-1 and epithelial phospho-Akt. Thus, loss of stromal Cav-1 is predictive of elevated levels of epithelial Cav-1 and epithelial Akt-activation. This provides important new clinical evidence for paracrine signaling between prostate cancer epithelial cells and the tumor stromal micro-environment, especially related to disease progression and metastasis.     

Characterization of cultured human prostatic epithelial cells by cluster designation antigen expression

Cultured prostatic epithelial cells have been extensively studied as a model of prostate biology. What is the lineage relationship of the cultured cells to the epithelial cell types in tissue? How different are cultured cells derived from tumor tissue to those derived from benign tissue? Expression of cluster designation (CD) cell surface molecules has been shown to be useful in characterizing cells according to lineage. A CD profile was therefore generated for cultured human prostatic epithelial cells and compared with those previously established for basal and luminal epithelial cells in the prostate. Presence of CD44, CD49b, CD49f, and CD104 and absence of CD57 suggests that cultured cells were derived from basal cells of prostatic tissues. However, expression of certain CD antigens characteristic of luminal epithelial cells was also observed in subpopulations of cultured cells. The pattern of CD antigens in cultured cells reflects a phenotype similar to that of transit-amplifying cells that have been described in the prostate. Several CD antigens were found expressed by both cultured prostatic epithelial and stromal cells, and are probably associated with cell proliferation. The CD profiles of cultured epithelial cell strains derived from normal compared with malignant tissues were notably similar to each other and to that of the prostate cancer cell line PC-3. We conclude that cells in culture retain expression of certain lineage-characteristic CD antigens. Furthermore, CD antigens can define subpopulations of cells with differential gene expression.     

Single-cell transcriptomics reveals cell type diversity of human prostate

Extensive studies have been performed to describe the phenotypic changes occurring during malignant transformation of the prostate. However, the cell types and associated changes that contribute to the development of prostate diseases and cancer remain elusive, largely due to the heterogeneous composition of prostatic tissues. Here, we conduct a comprehensive evaluation of four human prostate tissues by single-cell RNA sequencing (scRNA-seq) to analyze their cellular compositions. We identify 18 clusters of cell types, each with distinct gene expression profiles and unique features; of these, one cluster of epithelial cells (Ep) is found to be associated with immune function. In addition, we characterize a special cluster of fibroblasts and aberrant signaling changes associated with prostate cancer (PCa). Moreover, we provide insights into the epithelial changes that occur during the cellular senescence and aging. These results expand our understanding of the unique functional associations between the diverse prostatic cell types and the contributions of specific cell clusters to the malignant transformation of prostate tissues and PCa development.     

Expression of enzymes involved in estrogen metabolism in human prostate.

There is evidence that estrogens can directly modulate human prostate cell activity. It has also been shown that cultured human prostate cancer LNCaP can synthesize the active estrogen estradiol (E2). To elucidate the metabolism of estrogens in the human prostate, we have studied the expression of enzymes involved in the formation and inactivation of estrogens at the cellular level. 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 4, 7, and 12, as well as aromatase mRNA and protein expressions, were studied in benign prostatic hyperplasia (BPH) specimens using in situ hybridization and immunohistochemistry. For 17beta-HSD type 4, only in situ hybridization studies were performed. Identical results were obtained with in situ hybridization and immunohistochemistry. All the enzymes studied were shown to be expressed in both epithelial and stromal cells, with the exception of 17beta-HSD types 4 and 7, which were detected only in the epithelial cells. On the basis of our previous results, showing that 3beta-HSD and 17beta-HSD type 5 are expressed in human prostate, and of the present data, it can be concluded that the human prostate expresses all the enzymes involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2. The local biosynthesis of E2 might be involved in the development and/or progression of prostate pathology such as BPH and prostate cancer through modulation of estrogen receptors, which are also expressed in epithelial and stromal cells.     

TGF-β1 induces an age-dependent inflammation of nerve ganglia and fibroplasia in the prostate gland stroma of a novel transgenic mouse

TGF-β1 is overexpressed in wound repair and in most proliferative disorders including benign prostatic hyperplasia and prostate cancer. The stromal microenvironment at these sites is reactive and typified by altered phenotype, matrix deposition, inflammatory responses, and alterations in nerve density and biology. TGF-β1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-β1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-β1 via the enhanced probasin promoter to the murine prostate gland epithelium. Transgenic mice developed age-dependent lesions leading to severe, yet focal attenuation of epithelium, and a discontinuous basal lamina. These changes were associated with elevated fibroplasia and frequency of collagenous micronodules in collapsed acini, along with an induced inflammation in nerve ganglia and small vessels. Elevated recruitment of CD115+ myeloid cells but not mature macrophages was observed in nerve ganglia, also in an age-dependent manner. Similar phenotypic changes were observed using a human prostate epithelium tissue recombination xenograft model, where epithelial cells engineered to overexpress TGF-β1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-β1 expression induces a fibroplasia stromal response associated with breach of epithelial wall structure and inflammatory involvement of nerve ganglia and vessels. The novel findings of ganglia and vessel inflammation associated with formation of collagenous micronodules in collapsed acini is important as each of these are observed in human prostate carcinoma and may play a role in disease progression.     

EGF promotes the shedding of soluble E-cadherin in an ADAM10-dependent manner in prostate epithelial cells

During the progression of prostate cancer, the epithelial adhesion molecule E-cadherin is cleaved from the cell surface by ADAM15 proteolytic processing, generating an extracellular 80 kDa fragment referred to as soluble E-cadherin (sE-cad). Contrary to observations in cancer, the generation of sE-cad appears to correlate with ADAM10 activity in benign prostatic epithelium. The ADAM10-specific inhibitor INCB8765 and the ADAM10 prodomain inhibit the generation of sE-cad, as well as downstream signaling and cell proliferation. Addition of EGF or amphiregulin (AREG) to these untransformed cell lines increases the amount of sE-cad shed into the conditioned media, as well as sE-cad bound to EGFR. EGF-associated shedding appears to be mediated by ADAM10 as shRNA knockdown of ADAM10 results in reduced shedding of sE-cad. To examine the physiologic role of sE-cad on benign prostatic epithelium, we treated BPH-1 and large T immortalized prostate epithelial cells (PrEC) with an sE-cad chimera comprised of the human Fc domain of IgG₁, fused to the extracellular domains of E-cadherin (Fc-Ecad). The treatment of untransformed prostate epithelial cells with Fc-Ecad resulted in phosphorylation of EGFR and downstream signaling through ERK and increased cell proliferation. Pre-treating BPH-1 and PrEC cells with cetuximab, a therapeutic monoclonal antibody against EGFR, decreased the ability of Fc-Ecad to induce EGFR phosphorylation, downstream signaling, and proliferation. These data suggest that ADAM10-generated sE-cad may have a role in EGFR signaling independent of traditional EGFR ligands.     

Expression signature of the mouse prostate

Genetically engineered mice are being used increasingly for delineating the molecular mechanisms of prostate cancer development. Epithelium-stroma interactions play a critical role in prostate development and tumorigenesis. To better understand gene expression patterns in the normal sexually mature mouse prostate, epithelium and stroma were laser-capture microdissected from ventral, dorsolateral, and anterior prostate lobes. Genome-wide expression was measured by DNA microarrays. Our analysis indicated that the gene expression pattern in the mouse dorsolateral lobe was closest to that of the human prostate peripheral zone, supporting the hypothesis that these prostate compartments are functionally equivalent. Stroma from a given lobe had closer gene expression patterns with stroma from other lobes than epithelium from the same lobe. Stroma appeared to have higher expression complexity than epithelium. Specifically, stromal cells had higher expression levels of genes implicated in cell adhesion, muscle development, and contraction, in structural constituents of cytoskeleton and actin binding, and in components such as sarcomere and extracellular matrix collagen. Among the genes that were enriched in the epithelium were secretory proteins, including seminal vesicle protein secretion 2 and 5. Surprisingly, prostate stroma expressed many osteogenic molecules, as confirmed by immunohistochemistry. A "bone-like" environment in the prostate may predispose prostate cells for survival in the bone. Chemokine Cxcl12 but not its receptor, Cxcr4, was expressed in normal prostate. In prostate tumors, interestingly, Cxcl12 was up-regulated in epithelial cells with a concomitant expression of Cxcr4. Expression of both the receptor and ligand may provide an autocrine mechanism for tumor cell migration and invasion.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.     

Prostate epithelial stem cell culture

The prostate gland is the site of the second most common cancer in men in the UK, with 9,280 deaths recorded in 2000. Another common disease of the prostate is benign prostatic hyperplasia and both conditions are believed to arise as a result of changes in the balance between cell proliferation and differentiation. There are three types of prostatic epithelial cell, proliferative basal, secretory luminal, and neuroendocrine. All three are believed to be derived from a common stem cell through differentiation along different pathways but the mechanisms behind these processes is poorly understood. In particular, there has until recently been very little information about prostate stem cell growth and differentiation. This review will discuss ways of distinguishing these prostate cell types using markers, such as keratins. Methods available for the culture of prostate epithelial cells and for the characterisation of stem cells both in monolayer and three-dimensional models are examined. This revised version was published online in July 2006 with corrections to the Cover Date.