Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.     

雄激素受体对前列腺癌细胞侵袭转移能力的影响及其意义

目的研究雄激素受体（AR）对雄激素非依赖性前列腺癌细胞株PC3侵袭转移能力的影响及意义。方法应用肿瘤细胞侵袭实验、创伤愈合实验以及软琼脂糖凝胶实验检测AR对PC3侵袭转移能力的影响。结果外源性AR转染到PC3（PC3-AR）后,其侵袭能力明显减弱,创伤愈合能力降低,在琼脂糖凝胶中形成克隆能力降低。结论AR明显降低雄激素非依赖性PC3的侵袭转移能力。     

Prostate diseases--role of sex steroids and their inhibitors

The normal prostate as well as prostatic diseases are influenced by androgens. The exact reason for an altered and uncontrolled response to androgens, whether benign as in benign prostate hyperplasia (BPH) or malignant as in the case of prostate cancer (PC), is not known in detail. Nevertheless, restriction of androgen receptor activation by reduction of available androgens is of great clinical value in both diseases. In BPH the inhibition of the conversion of testosterone into 5α-dihydrotestosterone (DHT) by 5α-reductase (5AR) is highly efficient and used in general practice, while the situation in PC is more complex. Specific inhibition of 5AR does not provide as efficient relief of symptoms as general androgen deprivation therapy (ADT), and the use of 5ARI for PC prevention is still under debate. Further, the altered steroid metabolism in castration resistant prostate cancer (CRPC) together with the complex paracrine signalling between different androgen responsive cell types, make the development of more specific drugs targeting androgen receptor signalling both more relevant and challenging.     

Androgen-independent prostate cancer cells acquire the complete steroidogenic potential of synthesizing testosterone from cholesterol

The proliferation and differentiation of normal prostate epithelial cells depends upon the action of androgens produced by the testis. Prostate cancers retain the ability to respond to androgens in the initial stages of cancer development, but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining the expression of functional androgen receptor (AR). In the present study, we have determined the potential of prostate cancer cells to synthesize androgens from cholesterol which may be involved in intracrine regulation of AR in advanced stages of the disease. Established androgen-independent prostate cancer cell lines, PC3 and DU145 cells, expressed mRNA and proteins for scavenger receptor type B1 (SRB1), steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and other enzymes involved in androgen biosynthesis. Expression of all these proteins and enzymes was significantly higher in the androgen-independent derivative of LNCaP prostate cancer cells (C81) than in the androgen-dependent cell line (C33). In serum-free cultures, the androgen-independent C81 cells secreted approximately 5-fold higher testosterone than C33 cells as determined in the conditioned media by immunoassays. These cells could also directly convert radioactive cholesterol into testosterone which was identified by thin layer chromatography. These results for the first time show that prostate cancer cells in advanced stages of the disease could synthesize androgens from cholesterol and hence are not dependent upon testicular and/or adrenal androgens.     

Suppressor role of androgen receptor in proliferation of prostate basal epithelial and progenitor cells

Early studies have reported the differential roles of androgen receptor (AR) in different types (luminal, basal intermediate, and stromal) of prostate cancer cells. In vivo mouse model tumor studies using the total prostate epithelial knockout mice (pes-ARKO) also revealed that AR played a suppressive role in proliferation of the CK5(+)/CK8(+) progenitor/intermediate cells but a positive role in the CK5(-)/CK8(+) luminal epithelial cells. Using three different resources (one human basal epithelial cell line, one mouse basal epithelial originated progenitor cell line, and a basal epithelium-specific ARKO mouse model), we here demonstrated that the AR in basal epithelial cells of normal prostate plays a suppressive role in their proliferation but a positive role in differentiation into luminal epithelial cells. These results led us to conclude that ARs may play a negative role to suppress CK5(+) basal epithelial and progenitor cell proliferation, yet play an essential role to drive basal epithelial cells into more differentiated states. These results may explain why differential AR expression in different cell types within normal prostate is needed and suggest that ARs in prostate basal epithelial cells, although expressed at a very low level, are necessary to maintain the balance between progenitor cells and differentiated luminal epithelial cells.     

Disruption of prostate epithelial androgen receptor impedes prostate lobe-specific growth and function

Prostate development and maturation requires stromal-epithelial interactions and androgen action via the androgen receptor (AR) within these compartments. However, the specific roles of epithelial and stromal AR in postnatal prostate differentiation are unclear. We used Cre-LoxP technology to determine the prostate phenotype in mice with epithelial-selective genetic inactivation of the AR leaving the stromal AR functionally intact. We find that prostate development abolished in mice globally lacking a functional AR can be rescued by restricting the AR knockout to the postnatal prostate epithelium. We show that, at 8 wk of age, prostate epithelial AR knockout (PEARKO) mice exhibit prostate development with normal branching morphogenesis but lobe-specific decrease in prostate weight and hindered structural and functional differentiation of the mature prostate epithelium. No change was observed in PEARKO testis weight or serum testosterone compared with littermate controls. The most striking change was increased proliferation and abnormal lesions of epithelial cells predominantly in the anterior lobe of PEARKO mice. These findings highlight the vital role of stromal AR in postnatal prostate growth and structural differentiation and emphasize the requirement of epithelial AR in maintaining functional differentiation and restraining proliferation of epithelial cells in a lobe-specific manner. This unique PEARKO mouse provides a new paradigm with which to define the molecular mechanisms of the androgen signaling in mature prostate lobes in vivo and provides insight into the identification of better targets for treatment of prostate cancer and hyperplasia.     

Expression of GnRH type II is regulated by the androgen receptor in prostate cancer

GnRH II has important functional effects in steroid hormone-dependent tumours. Here we investigated the expression and regulation of GnRH II in prostate cancer. GnRH II protein was equally expressed in benign (73%) and malignant (78%) biopsies studied in a prostate tissue microarray (P = 0.779). There was no relationship between expression and clinical parameters in the cancer cohort. GnRH II was, however, significantly reduced in tumour biopsies following hormone ablation. This was further investigated in a prostate xenograft model where androgens increased GnRH II levels, while their withdrawal reduced it. In cell lines, we confirmed high levels of GnRH II in androgen receptor (AR)-positive LNCaP cells but low levels in AR-negative PC3 cells. In LNCaP cells, GnRH II induction by androgens was blocked by the AR inhibitor casodex, but not by cycloheximide treatment. Sequence analysis subsequently revealed a putative androgen response element in the upstream region of the GnRH II gene and direct interaction with the AR was confirmed in chromatin immunoprecipitation experiments. Finally, to test whether the effects of GnRH II were dependent on AR expression, LNCaP and PC3 cells were exposed to exogenous peptide. In both cell lines, GnRH II inhibited cell proliferation and migration, suggesting that its function is independent of AR status. These results provide evidence that GnRH II is widely expressed in prostate cancer and is an AR-regulated gene. Further studies are warranted to characterise the effects of GnRH II on prostate cancer cells and investigate its potential value as a novel therapy.     

Androgen receptor expression in prostate carcinoma cells suppresses alpha6beta4 integrin-mediated invasive phenotype

Prostate cancer cells may lose androgen-sensitivity after androgen ablation therapy, becoming highly invasive and metastatic. The biological mechanisms responsible for higher tumurogenicity of androgen-independent prostate carcinomas are not entirely known. We demonstrate that androgen receptor regulation of adhesion and invasion of prostate cancer cells through modulation of alpha6beta4 integrin expression may be one of the molecular mechanisms responsible of this phenomenon. We found that protein and gene expressions of alpha6 and beta4 subunits were strongly reduced in the androgen-sensitive cell line LNCaP respect to the androgen-independent PC3 and that transfection of PC3 cells with a full-length androgen receptor expression vector resulted in a decreased expression of alpha6beta4 integrin, reduced adhesion on laminin, and suppressed Matrigel invasion. Growth in soft agar was also suppressed in androgen receptor-positive PC3 clones. Treatment of androgen receptor positive clones with the synthetic androgen R1881 further reduced alpha6 and beta4 messenger RNA expression as well as adhesion on laminin and Matrigel invasion. Our results indicate that androgens regulate cell-extracellular matrix adhesion and invasion by modulation of integrin expression and function, thus keeping a low invasive phenotype of prostate cancer cells.     

Identification and characterization of an androgen-responsive gene encoding an aci-reductone dioxygenase-like protein in the rat prostate

The ALP1 [aci-reductone dioxygenase (ARD)-like protein 1] gene was identified in a comprehensive cDNA subtraction aimed at identifying genes regulated by androgens in the rat ventral prostate. ALP1 is homologous to the ARD/ARD' that were discovered in Klebsiella pneumoniae as enzymes that have the same polypeptide sequence and differ only in their metal content. This family of proteins is evolutionarily conserved from bacteria to humans and is involved in the methionine salvage pathway. Northern and Western blot confirmed the regulation of ALP1 by androgens in the rat ventral prostate. ALP1 mRNA is expressed in a variety of tissues; however, its regulation by androgens was specific to the prostate. ALP1 is expressed by the glandular epithelial cells of the rat prostate, with little or no expression in the stromal cells. ALP1 is down-regulated in the different rat Dunning tumor cell lines compared with the normal or castrated rat prostate. Expression studies showed that ALP1 overexpression is not tolerated by AT6.1 cells. Further studies demonstrated that ALP1 is also down-regulated in the human prostate cancer cell lines LNCaP, PC3, and DU145, and overexpression induces cell death in these cells. Taken together, our observations suggest that ALP1 may have an important role in androgen regulated prostate homeostasis as well as in prostate cancer progression by regulating cell death of prostate cancer cells.     

Altered endocytosis of epidermal growth factor receptor in androgen receptor positive prostate cancer cell lines

Although androgens and the androgen receptor (AR) are involved in tumorigenesis of prostate cancer (PC) in initial phases, less clear is the role played in advanced androgen-independent (AI) stages of the disease. Several recent reports indicated that re-expression of AR in PC-derived cell lines determines a less aggressive phenotype of the cells. We have previously demonstrated that re-expression of AR decreases the invasion ability of PC3 cells in vitro by affecting signalling and internalization processes of epidermal growth factor receptor (EGFR). Here, we show that reduced EGFR internalization is also a characteristic of AR positive PC cell lines LNCaP and 22Rv1. Reduced internalization in PC3-AR cells is associated to a defective interaction between the EGFR and two adaptor proteins which mediate the endocytotic process, Grb2 and c-Cbl. As a consequence of such reduced interaction, ubiquitination of the receptor, which is mainly mediated by c-Cbl, is also altered. In addition, we show that internalized EGFR co-localizes with early endosome antigen-1, a marker of clathrin-mediated endocytosis, in PC3-Neo cells but not in AR positive cell lines. Conversely, EGFR maintains co-localization with caveolin-1 after EGF stimulation in PC3-AR cells. These data suggest that expression of AR affects clathrin-mediated endocytosis pathway of EGFR, which, according to recent findings, plays an essential role in the completeness of signalling of the receptor. Taken together, these data emphasize the role of AR in the regulation of EGFR endocytotic trafficking and active signalling in PC cells. In view of the role of EGFR signalling in invasion of carcinoma cells, our data may explain the lower invasive phenotype observed in AR-positive cell lines.     

Androgens and esophageal cancer: What do we know?

Significant disparities exist between genders for the development and progression of several gastro-intestinal (GI) diseases including cancer. Differences in incidence between men vs women for colon, gastric and hepatocellular cancers suggest a role for steroid sex hormones in regulation of GI carcinogenesis. Involvement of intrinsic gender-linked mechanisms is also possible for esophageal adenocarcinoma as its incidence is disproportionally high among men. However, the cause of the observed gender differences and the potential role of androgens in esophageal carcinogenesis remains unclear, even though the cancer-promoting role of androgen receptors (AR) shown in other cancers such as prostate and bladder suggests this aspect warrants exploration. Several studies have demonstrated expression of ARs in esophageal cancer. However, only one study has suggested a potential link between AR signaling and outcome - poorer prognosis. Two groups have analyzed data from cohorts with prostate cancer and one of these found a decreased incidence of esophageal squamous and adenocarcinoma after androgen deprivation therapy. However, very limited information is available about the effects of androgen and AR-initiated signaling on esophageal cancer cell growth in vitro and in vivo. Possible mechanisms for androgens/AR involvement in the regulation of esophageal cancer growth are considered, and the potential use of AR as a prognostic factor and clinical target is highlighted, although insufficient evidence is available to support clinical trials of novel therapies. As esophageal adenocarcinoma is a gender linked cancer with a large male predominance further studies are warranted to clarify the role of androgens and ARs in shaping intracellular signaling and genomic responses in esophageal cancer.     

Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression.

Brief administration of estrogen to newborn rats results in permanent suppression of prostate growth and reduced prostatic responsiveness to testosterone in adulthood. To determine whether this imprinting may be a result of alterations in androgen receptor (AR) expression, the separate adult prostate lobes of neonatally estrogenized rats were examined for AR concentration and distribution. Sprague-Dawley rat pups were given 25 micrograms estradiol benzoate or oil alone on days 1, 3, and 5 and were killed on day 90. Half of the animals received 2-cm testosterone implants 10 days before death to assess the activational response to androgen. In a separate series, neonatally estrogenized rats were given prepubertal dihydrotestosterone pellets for 3 weeks as well as testosterone implants in adulthood to determine if the observed effects of neonatal estrogen on the adult prostate were an indirect result of androgen deprivation during developmentally critical periods. The ventral, dorsal, and lateral prostate lobes were processed for nuclear AR quantitation by [3H]dihydrotestosterone exchange binding assay and for indirect immunocytochemical localization of AR. Weights and DNA contents of the three prostate lobes were significantly reduced in neonatally estrogenized rats, and this decrease was only partially reversed by prepubertal and/or adult androgen replacement. Histologically, the hypoplastic ventral and dorsal lobes exhibited a relative increase in interacinar stromal tissue, disorganized acini with epithelial hyperplasia, luminal sloughing, and an apparent lack of differentiation. The hypoplastic lateral lobe also showed a relative increase in the stromal fraction; however, the acinar epithelium appeared differentiated, with normal basal/apical orientation and luminal secretions. The AR concentration was significantly reduced in the ventral and dorsal prostates of estrogenized rats, but was unaltered in the lateral lobe. Immunocytochemistry revealed a marked reduction or absence of epithelial AR in ventral and dorsal lobes from estrogenized rats, whereas the lateral lobe epithelial cells expressed AR similarly to controls. The incidence of AR-positive fibroblastic stromal cells increased in lateral prostates from 5% in controls to approximately 25% in estrogenized rats. Neonatally estrogenized rats given testosterone for 10 days in adulthood showed increased levels of AR in the ventral and dorsal lobes compared to nonstimulated rats; however, these levels remained well below control values. Lateral lobe epithelial histology and AR expression appeared relatively unchanged in estrogenized rats given testosterone during adulthood, whereas an increased proportion of stromal cells (approximately 35%) were AR positive. In summary, neonatal estrogen administration permanently altered prostatic growth and produced lobe-specific changes in AR expression in the adult gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression.

Brief administration of estrogen to newborn rats results in permanent suppression of prostate growth and reduced prostatic responsiveness to testosterone in adulthood. To determine whether this imprinting may be a result of alterations in androgen receptor (AR) expression, the separate adult prostate lobes of neonatally estrogenized rats were examined for AR concentration and distribution. Sprague-Dawley rat pups were given 25 micrograms estradiol benzoate or oil alone on days 1, 3, and 5 and were killed on day 90. Half of the animals received 2-cm testosterone implants 10 days before death to assess the activational response to androgen. In a separate series, neonatally estrogenized rats were given prepubertal dihydrotestosterone pellets for 3 weeks as well as testosterone implants in adulthood to determine if the observed effects of neonatal estrogen on the adult prostate were an indirect result of androgen deprivation during developmentally critical periods. The ventral, dorsal, and lateral prostate lobes were processed for nuclear AR quantitation by [3H]dihydrotestosterone exchange binding assay and for indirect immunocytochemical localization of AR. Weights and DNA contents of the three prostate lobes were significantly reduced in neonatally estrogenized rats, and this decrease was only partially reversed by prepubertal and/or adult androgen replacement. Histologically, the hypoplastic ventral and dorsal lobes exhibited a relative increase in interacinar stromal tissue, disorganized acini with epithelial hyperplasia, luminal sloughing, and an apparent lack of differentiation. The hypoplastic lateral lobe also showed a relative increase in the stromal fraction; however, the acinar epithelium appeared differentiated, with normal basal/apical orientation and luminal secretions. The AR concentration was significantly reduced in the ventral and dorsal prostates of estrogenized rats, but was unaltered in the lateral lobe. Immunocytochemistry revealed a marked reduction or absence of epithelial AR in ventral and dorsal lobes from estrogenized rats, whereas the lateral lobe epithelial cells expressed AR similarly to controls. The incidence of AR-positive fibroblastic stromal cells increased in lateral prostates from 5% in controls to approximately 25% in estrogenized rats. Neonatally estrogenized rats given testosterone for 10 days in adulthood showed increased levels of AR in the ventral and dorsal lobes compared to nonstimulated rats; however, these levels remained well below control values. Lateral lobe epithelial histology and AR expression appeared relatively unchanged in estrogenized rats given testosterone during adulthood, whereas an increased proportion of stromal cells (approximately 35%) were AR positive. In summary, neonatal estrogen administration permanently altered prostatic growth and produced lobe-specific changes in AR expression in the adult gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gadd45gamma is androgen-responsive and growth-inhibitory in prostate cancer cells

In our previous microarray analysis searching for genes differentially regulated by androgens in the rat ventral prostate, we identified GADD45gamma (growth arrest and DNA damage inducible, gamma) as one of the genes up-regulated by androgens. GADD45gamma was initially identified to be a gene involved in negative growth control and its overexpression induced cycle arrest and apoptosis in vitro. In this study, we showed that GADD45gamma was transiently up-regulated by androgens in the androgen-responsive human prostate cancer cell line LNCaP. The GADD45gamma up-regulation was blocked by an androgen receptor (AR) antagonist, bicalutamide, suggesting the involvement of the androgen receptor. However, this up-regulation was inhibited by cycloheximide, indicating that GADD45gamma induction by androgens requires new protein synthesis. Overexpression of GADD45gamma inhibited cell growth of LNCaP and PC3 cells and resulted in dramatic morphological changes in both cell lines, arguing that GADD45gamma is likely to participate in the differentiation program induced by androgens in the prostate. The above observations provide evidence that GADD45gamma is an androgen-responsive gene with growth-inhibitory activity in human prostate cancer cells.     

Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs

There is now evidence that oestrogens and androgens can influence male and female reproductive systems. In order to accurately identify the sites of action of oestrogens and androgens, we have proceeded to the histological localization of the two oestrogen receptor (ER) subtypes, ERalpha and ERbeta, and the androgen receptor (AR) in the reproductive tissues of adult rats of both sexes. AR was detected by immunocytochemistry, while ERalpha and ERbeta were localized by both immunocytochemistry and in situ hybridization. In the pituitary gland of animals of both sexes, ERalpha was found in the majority of nuclei of secretory cells in the anterior pituitary. The intermediate and posterior lobes did not show any staining. ERbeta was not found to be expressed in any of the pituitary lobes. Using AR antibodies, nuclear staining was detected in about 50% of secretory cells of the anterior lobe, the intermediate and posterior lobes being completely unstained. In the testis, ERalpha was localized in nuclei of Leydig cells as well as in round spermatocytes and spermatids, while ERbeta could only be detected in Sertoli cell nuclei. AR immunoreactivity was found in nuclei of Sertoli, peritubular myoid and Leydig cells. In the prostate, ERbeta was observed in epithelial cells of tubulo-alveoli, while the stroma was unlabelled. ERalpha was not found to be expressed in any prostate cells. In the prostate, AR was detected in nuclei of epithelial, stromal and endothelial cells. In seminal vesicles, staining of ERalpha was found in nuclei of epithelial and stromal cells. Similar findings were observed using AR antibodies. While ERbeta mRNA could not be detected by in situ hybridization, weak staining for ERbeta was localized in epithelial cells of seminal vesicles. In the ovary, both ERalpha and ERbeta were found to be expressed. ERbeta mRNA was found in granulosa cells of growing follicles, while ERalpha was present in theca cells, interstitial gland cells and germinal epithelium. AR immunoreactivity was detected in granulosa cell nuclei in growing follicles and also in scattered interstitial cells. In the oviduct and uterus, ERalpha was observed in nuclei of epithelial cells as well as of stromal and muscle cells. Similarly, AR immunoreactivity was present in nuclei of epithelial cells, stromal and muscle cells in both the oviduct and uterus. ERbeta was not detected in the oviduct and uterus. The present findings indicate a cell-specific localization of ERalpha, ERbeta and AR in reproductive tissues in rats of both sexes. By establishing the precise sites of action of oestrogens and androgens they contribute to a better understanding of the respective role of these steroids in reproduction function.     

Androgens regulate the immune/inflammatory response and cell survival pathways in rat ventral prostate epithelial cells

A major hurdle in understanding the role of androgens is the heterogeneity of androgen receptor (AR) expression in the prostate. Because the majority of prostate cancer arises from the AR-positive secretory luminal epithelial cells, identifying the androgen-mediated pathways in the prostate epithelium is of great significance to understanding their role in prostate pathogenesis. To meet this objective, the current study was designed to identify immediate-early genes expressed in response to the synthetic androgen R1881 in cultured rat ventral prostate epithelial cells. Rat ventral prostate epithelial cells, purified from 20-d-old rats, were cultured, and the presence of AR and the response to androgen were established. The cells were then treated with R1881 for 2 and 12 h to capture immediate-early genes in an Affymetrix-based gene chip platform. A total of 66 nonredundant genes were identified that were responsive to R1881. The functional androgen response elements were identified in the proximal promoter to determine possible molecular mechanism. Cluster analysis identified five distinct signatures of R1881-induced genes. Pathway analysis suggested that R1881 primarily influences cell proliferation/differentiation and inflammatory/immune response pathways. Androgens appear to regulate cell renewal by regulating differentiation, cell proliferation, and apoptosis. Two mutually exclusive inflammatory response pathways were observed. The interferon pathway was up-regulated, and the ILs were down-regulated. The data identified novel androgen-regulated genes (e.g. Id1, Id3, IL-6, IGF-binding protein-2 and -3, and JunB). The loss of androgen regulation of these genes can have important consequences for cellular transformation and transition to androgen-independent growth and survival.