Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.     

Differential expression of the mouse mitochondrial genes and the mitochondrial RNA-processing endoribonuclease RNA by androgens.

Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.     

MicroRNAs are mediators of androgen action in prostate and muscle

Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3' UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues.     

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.     

Autoregulation of androgen receptor expression in rodent prostate: immunohistochemical and in situ hybridization analysis

Autoregulation of androgen receptor mRNA and protein was investigated by immunohistochemical and in situ hybridization techniques. In both mouse and rat prostate, the epithelial cell nuclei were stained with the monoclonal or polyclonal antibodies raised against human androgen receptor. It was observed that 3 days after castration, nuclear staining of the epithelium was greatly reduced, while androgen treatment restored the staining intensity to a normal level. In situ hybridization using an androgen receptor cDNA fragment as probe demonstrated that the change in androgen receptor mRNA level correlated with the change in antibody staining intensity. These data suggested an up-regulation of androgen receptor expression by androgen.     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.     

Notch1-expressing cells are indispensable for prostatic branching morphogenesis during development and re-growth following castration and androgen replacement

Notch expression is frequently associated with progenitor cells, and its function is crucial for development. Our recent work showing that Notch1 is selectively expressed in basal epithelial cells of the prostate and higher Notch1 expression during development suggests that Notch1-expressing cells may define progenitor cells in the prostate. To test this hypothesis, we have generated a transgenic mouse line in which the Notch1-expressing cells can be ablated in a controlled manner. Specific targeting was achieved by expressing the bacterial nitroreductase, an enzyme that catalyzes its substrate into a cytotoxin capable of inducing apoptosis, under the Notch1 promoter. Cell death in transgenic prostate was confirmed by histological analyses including terminal dUTP nick-end labeling and caspase 3 immunocytochemical staining. We evaluated the consequences of ablation of Notch1-expressing cells in two systems, organ culture of early postnatal prostates and re-growth of prostate in castrated mice triggered by hormone replacement. Our data show that elimination of Notch1-expressing cells inhibited the branching morphogenesis, growth, and differentiation of early postnatal prostate in culture and impaired prostate re-growth triggered by hormone replacement in castrated mice. Furthermore, we found that Notch1 expression following castration and hormone replacement was concomitant with known basal cell markers p63 and cytokeratin 14 and was high in the proliferative human prostate epithelial cells. Taken together, these data suggest that Notch1-expressing cells define the progenitor cells in the prostatic epithelial cell lineage, which are indispensable for prostatic development and re-growth.     

A transcriptional target of androgen receptor,miR-421 regulates proliferation and metabolism of prostate cancer cells

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.     

Surgical Stress Delays Prostate Involution in Mice

Androgens control growth of prostate epithelial cells and androgen deprivation induces apoptosis, leading to prostate involution. We investigated the effects of surgical stress on prostate involution induced by androgen ablation and determined the underlying mechanisms. Androgen ablation in mice was induced by surgical castration and administration of the anti-androgenic drugs bicalutamide and MDV3100. Surgical stress was induced by sham castration under isoflurane anesthesia. Surgical stress delayed apoptosis and prostate involution induced by anti-androgenic drugs. These effects of stress were prevented by administering the selective beta2-adrenoreceptor antagonist ICI118,551 and were also blocked in BAD^3SA/WT mice expressing phosphorylation-deficient mutant BAD3SA. These results indicate that apoptosis and prostate involution in response to androgen ablation therapy could be delayed by surgical stress via the beta2-adrenoreceptor/BAD signaling pathway. Thus, surgery could interfere with androgen ablation therapy, whereas administration of beta2-adrenoreceptor antagonists may enhance its efficacy.     

The Notch pathway in prostate development and cancer

Abstract The Notch family of transmembrane receptors are important mediators of cell fate determination. Accordingly, Notch signaling is intimately involved in the development of numerous tissues. Recent findings have highlighted a critical role for Notch signaling in normal prostate development. Notch signaling is required for embryonic and postnatal prostatic growth and development, for proper cell lineage specification within the prostate, as well as for adult prostate maintenance and regeneration following castration and hormone replacement. Evidence for Notch as a regulator of prostate cancer development, progression, and metastasis has also emerged. This review summarizes our current understanding of the role of Notch pathway elements, including members of the Jagged, Delta-like, hairy/enhancer-of-split, and hairy/enhancer-of-split related with YRPW motif families, in prostate development and tumorigenesis. Data supporting Notch pathway elements as oncogenes and tumor suppressors in prostate tumors, as well as data implicating Notch receptors and ligands as potential markers of normal prostate stem/progenitor cells and prostate cancer stem/initiating cells, are also presented.     

Hormone-independent polyamine metabolism of squamous cell carcinoma of the prostate

The levels of polyamines and their synthesizing enzymes in squamous cell carcinoma of prostate implanted in intact as well as castrated male rats were determined after certain hormonal manipulations. The tumour was found to grow with an identical rate in non-castrated and castrated rats. Polyamine content and activities of polyamine synthesizing enzymes in the tumour were found to be much lower compared to their values in ventral prostate. Moreover, the levels of these parameters were comparable in tumours whether implanted in non-castrated or gonadectomized animals. The sequential analyses of putrescine and spermidine and activities of L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase of tumours at different time intervals showed a significant reduction in their levels at 30 days compared to 10 days post implantation in non-castrated as well as castrated rats. Daily intramuscular administration of tumour-bearing intact or castrated animals with testosterone (50 micrograms/g), beta-estradiol (2 micrograms/g) or cyproterone (12.5 micrograms/g) for 10 days did not influence polyamine metabolism in tumour tissue. However, either beta-estradiol and cyproterone treatments or castration were found to decrease polyamine synthesis in ventral prostate. At the same time, the testosterone replacement therapy did not allow polyamine levels or activities of polyamine synthesizing enzymes to decline in the ventral prostate of castrated rats. Our results demonstrated that contrary to ventral prostate, the polyamine metabolism in squamous cell carcinoma of prostate is independent of hormonal control. The loss of hormonal sensitivity of polyamine metabolism in the prostatic tumour could be the result of qualitative changes that occurred during transformation.     

Caveolin-1, a metastasis-related gene that promotes cell survival in prostate cancer

Metastasis represents the ultimate target in cancer therapy as this complex biological process is the direct cause of mortality for a variety of human malignancies. The current high level of mortality from prostate cancer results in large part from the inexorable growth of overt or occult metastasis present at the time of diagnosis. Currently, there are no curative therapies for metastatic prostate cancer. To better understand the metastatic phenotype in prostate cancer, we developed a strategy to identify mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer using differential display-PCR. In using this system a number of metastasis-related sequences were identified including a cDNA that encodes caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also in human metastatic disease. Recent studies have indicated that suppression of caveolin-1 expression induces androgen sensitivity in high caveolin-1, androgen-insensitive mouse prostate cancer cells derived from metastases. Conversely, overexpression of caveolin-1 leads to androgen insensitivity in low caveolin, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is both a metastasis-related gene as well as a candidate androgen resistance gene for prostate cancer in man. Interestingly, recent studies also point to a potential role for caveolin-1 in the resistance of various malignancies to multiple antineoplastic agents. The linkage of caveolin-1 expression with the androgen-resistant phenotype in prostate cancer and the multidrug resistance phenotype in various solid tumors establishes a novel paradigm for understanding these clinically important and now potentially related processes in malignant progression.