WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear Β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector Β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.Peer reviewe

Modulation of the cytosolic androgen receptor in striated muscle by sex steroids

The influence of orchiectomy (GDX) and steroid administration on the level of the cytosolic androgen receptor in the rat levator ani muscle and in rat skeletal muscles (tibialis anterior and extensor digitorum longus) was studied. Androgen receptor binding to muscle cytosol was measured using H-3 methyltrienolone (R1881) as ligand, 100 fold molar excess unlabeled R1881 to assess nonspecific binding, and 500 fold molar excess of triamcinolone acetonide to prevent binding to glucocorticoid and progestin receptors. Results demonstrate that modification of the levels of sex steroids can alter the content of androgen receptors of rat striated muscle. Data suggest that: (1) cytosolic androgen receptor levels increase after orchiectomy in both levator ani muscle and skeletal muscle; (2) the acute increase in receptor levels is blocked by an inhibitor of protein synthesis; and (3) administration of estradiol-17 beta to castrated animals increases receptor binding in levator ani muscle but not in skeletal muscle

lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs.

Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy. Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In 'resistant' prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo. Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nht of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease Sl analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcrip tion 98 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to ceils results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in viva, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression

The androgen receptor and signal-transduction pathways in hormone-refractory prostate cancer. Part 1: modifications to the androgen receptor

Prostate cancer is the second most common male malignancy in the western world an increasing incidence in an ageing population. Treatment of advanced prostate cancer relies on androgen deprivation. Although the majority of patients initially respond favourably to androgen deprivation therapy, the mean time to relapse is 12-18 months. Currently there are few treatments available for men who have developed resistance to hormone therapy, due to the lack of understanding of the molecular mechanisms underlying development of this disease. Recently, however, major advances have been made in understanding both androgen receptor (AR) dependent and independent pathways which promote development of hormone resistant prostate cancer. This review will focus on modifications to the AR and associated pathways. Molecular modifications to the androgen receptor itself, e.g. mutations and/or amplification, although involved in the development of hormone resistance cannot explain all cases. Phosphorylation of AR, via either Ras/MAP kinase or PI3K/Akt signal transduction pathways, have been shown to activate AR in both a ligand (androgen) dependent and independent fashion. During this review we will discuss the clinical evidence to support AR dependent pathways as mediators of hormone resistance

The CAG trinucleotide repeat length in the androgen receptor does not predict the early onset of prostate cancer

Objective To relate the repeat length of the androgen-receptor CAG trinucleotide to the age of onset of prostate cancer, stage and grade of disease. Patients and methods After obtaining ethical approval, 265 patients with locally confined or locally advanced/metastatic prostate cancer were identified and evaluated for age at diagnosis (less than 65 years and greater than 75 years). DNA was extracted from peripheral blood lymphocytes and 1 mug aliquots subjected to polymerase chain reaction using fluorescently labelled primers. Samples were then run on an ABI 377 gene scan analysis gel with an internal molecular weight marker. The length of the CAG repeat was determined by comparing the gene scan product size to samples where the CAG repeat length had been quantified using direct sequencing. The Kruskal-Wallis, Mann-Whitney and Wilcoxon two sample tests were used to analyse the data. Results The mean (range) length of the CAG repeat in the androgen receptor was 22.2 (10-31) in the younger and 22.5 (16-32) in the older group, and was not statistically different. There was no significant association between the CAG repeat length and the age of onset of prostate cancer (P = 0.568) or with stage (P = 0.577) and grade (P = 0.891) of prostate cancer. Conclusion These results suggest that there is no correlation between the androgen receptor CAG repeat length and the age of onset, stage and grade of prostate cancer, confirming recent doubts from other similar studies of a suggested correlation between shorter androgen receptor CAG repeat and early onset and aggressiveness of prostate cancer

Renal Subcapsular xenografing of human fetal external genital tissue - A new model for investigating urethral development.

In this paper, we introduce our novel renal subcapsular xenograft model for the study of human penile urethral and clitoral development. We grafted fifteen intact fetal penes and clitorides 8-11 weeks fetal age under the renal capsules of gonadectomized athymic mice. The mice were treated with a subcutaneous pellet of dihydrotestosterone (DHT), diethylstilbestrol (DES) or untreated with hormones. Xenografts were harvested after fourteen days of growth and analyzed via serial histologic sectioning and immunostaining for Ki-67, cytokeratins 6, 7 and 10, uroplakin and the androgen receptor. Non-grafted specimens of similar fetal age were sectioned and immunostained for the same antigenic markers. 14/15 (93.3%) grafts were successfully propagated and harvested. The developing urethral plate, urethral groove, tubular urethra, corporal bodies and preputial lamina were easily identifiable. These structures demonstrated robust cellularity, appropriate architecture and abundant Ki-67 expression. Expression patterns of cytokeratins 6, 7 and 10, uroplakin and the androgen receptor in xenografted specimens demonstrated characteristic male/female differences analogous to non-grafted specimens. DHT treatment reliably produced tubularization of nascent urethral and vestibular structures and male patterns of androgen receptor expression in grafts of both genetic sexes while estrogenic or hormonally absent conditions reliably resulted in a persistent open urethral/vestibular groove and female patterns of androgen receptor expression. This model's success enables further study into causal pathways by which endocrine-disrupting and endocrine-mimicking substances may directly cause disruption of normal human urethral development or hypospadias

The effect of different types of hepatic injury on the estrogen and androgen receptor activity of liver

Mammalian liver contains receptors for both estrogens and androgens. Hepatic regeneration after partial hepatectomy in male rats is associated with a loss of certain male-specific hepatic characteristics. In this study we investigated the effects of lesser forms of hepatic injury on the levels of estrogen and androgen receptor activity in the liver. Adult male rats were subjected to portacaval shunt, partial portal vein ligation, hepatic artery ligation, or two-thirds partial hepatectomy. Another group of animals was treated with cyclosporine. At the time of sacrifice the livers were removed and used to determine the estrogen and androgen receptor activity in the hepatic cytosol. A significant reduction (p < 0.05) in the hepatic cytosolic androgen receptor activity and a slight increase in the estrogen receptor activity occurred following total portosystemic shunting. Partial ligation of the portal vein, which produces a lesser degree of portosystemic shunting, had no effect on the levels of the estrogen and androgen receptor activity present within hepatic cytosol. Cyclosporine-treated animals had significantly greater (p < 0.01) levels of estrogen receptor activity in the hepatic cytosol compared to vehicle-treated control animals. Levels of estrogen and androgen receptor activity within the hepatic cytosol remained unchanged after ligation of the hepatic artery. The reduction in the cytosolic estrogen and androgen receptor activity in the liver after partial hepatectomy was confirmed. In summary, certain types of hepatic injury are associated with profound changes in the estrogen and androgen receptor content within the liver. © 1989 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines

Abstract The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT coVon encoding a threonine residue to GCT, encoding alanine