Androgen-repressed phenotype in human prostate cancer

An androgen-repressed human prostate cancer cell line, ARCaP, was established and characterized. This cell line was derived from the ascites fluid of a patient with advanced metastatic disease. In contrast to the behavior of androgen-dependent LNCaP and its androgen-independent C4-2 subline, androgen and estrogen suppress the growth of ARCaP cells in a dose-dependent manner in vivo and in vitro. ARCaP is tumorigenic and highly metastatic. It metastasizes to the lymph node, lung, pancreas, liver, kidney, and bone, and forms ascites fluid in athymic hosts. ARCaP cells express low levels of androgen receptor mRNA and prostate-specific antigen mRNA and protein. Immunohistochemical staining shows that ARCaP cells stain intensely for epidermal growth factor receptor, c-erb B2/neu, and c-erb B3. Staining is negative for chromogranin A and positive for bombesin, serotonin, neuron-specific enolase, and the c-met protooncogene (a hepatic growth factor/scatter factor receptor). ARCaP cells also secrete high levels of gelatinase A and B and some stromelysin, which suggests that this cell line may contain markers representing invasive adenocarcinoma with selective neuronendocrine phenotypes. Along with its repression of growth, androgen is also found to repress the expression of prostate-specific antigen in ARCaP cells as detected by a prostate-specific antigen promoter–β-galactosidase reporter assay. Our results suggest that the androgen-repressed state may be central to prostate cancer progression and that advanced prostate cancer can progress from an androgen-independent to an androgen-repressed state.

Androgen-induced proliferative quiescence in prostate cancer cells: The role of AS3 as its mediator

In the prostate gland of adult mammals, most epithelial cells are in a state of proliferative quiescence. Androgens regulate this effect by inducing cell cycle arrest in the G0/G1 phase. Potential mediators of this androgen-induced proliferative shutoff were identified by means of subtracted cDNA libraries. The expression pattern of one of these sequences, AS3, strongly correlated with the expression of the androgen-induced proliferative shutoff both temporally and dosewise. The AS3 gene is located on chromosome 13 q12.3, in close proximity to the BRCA2 gene. The loss of chromosomal regions where AS3 alleles are located correlates with various human cancers, including prostate. The biological effect of AS3 was tested in two stable cell lines, one expressing sense and another expressing antisense AS3 constructs, both under tetracycline regulation. S9 cells were obtained by retroviral infection with virions containing a tetracycline-regulated sense AS3 construct. In these cells, sense AS3 was negatively regulated by tetracycline. Tetracycline withdrawal increased the expression of AS3 mRNA and protein. The expression of tetracycline-regulated AS3 resulted in inhibition of cell proliferation. A4 cells were obtained by retroviral infection with virions containing a tetracycline-regulated antisense AS3 construct. Vector-driven expression of antisense-AS3 blocked the induction of androgen-induced endogenous AS3 mRNA and blocked the inhibitory effect of androgens on cell proliferation. Tetracycline-regulated expression of the empty vector control had no effect on cell proliferation. These experiments strongly suggest that AS3 is a mediator of the androgen-induced proliferative shutoff.

Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: Role of vascular endothelial growth factor

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone–ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone–ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.

From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells

Although transforming growth factor-β (TGF-β) has been identified to mainly inhibit cell growth, the correlation of elevated TGF-β with increasing serum prostate-specific antigen (PSA) levels in metastatic stages of prostate cancer has also been well documented. The molecular mechanism for these two contrasting effects of TGF-β, however, remains unclear. Here we report that Smad3, a downstream mediator of the TGF-β signaling pathway, functions as a coregulator to enhance androgen receptor (AR)-mediated transactivation. Compared with the wild-type AR, Smad3 acts as a strong coregulator in the presence of 1 nM 5α-dihydrotestosterone, 10 nM 17β-estradiol, or 1 μM hydroxyflutamide for the LNCaP mutant AR (mtAR T877A), found in many prostate tumor patients. We further showed that endogenous PSA expression in LNCaP cells can be induced by 5α-dihydrotestosterone, and the addition of the Smad3 further induces PSA expression. Together, our findings establish Smad3 as an important coregulator for the androgen-signaling pathway and provide a possible explanation for the positive role of TGF-β in androgen-promoted prostate cancer growth.

Hyposecretion of the adrenal androgen dehydroepiandrosterone sulfate and its relation to clinical variables in inflammatory arthritis

Hypothalamic–pituitary–adrenal underactivity has been reported in rheumatoid arthritis (RA). This phenomenon has implications with regard to the pathogenesis and treatment of the disease. The present study was designed to evaluate the secretion of the adrenal androgen dehydroepiandrosterone sulfate (DHEAS) and its relation to clinical variables in RA, spondyloarthropathy (Spa), and undifferentiated inflammatory arthritis (UIA). Eighty-seven patients (38 with RA, 29 with Spa, and 20 with UIA) were studied, of whom 54 were women. Only 12 patients (14%) had taken glucocorticoids previously. Age-matched, healthy women (134) and men (149) served as controls. Fasting blood samples were taken for determination of the erythrocyte sedimentation rate (ESR), serum DHEAS and insulin, and plasma glucose. Insulin resistance was estimated by the homeostasis-model assessment (HOMAIR). DHEAS concentrations were significantly decreased in both women and men with inflammatory arthritis (IA) (P < 0.001). In 24 patients (28%), DHEAS levels were below the lower extreme ranges found for controls. Multiple intergroup comparisons revealed similarly decreased concentrations in each disease subset in both women and men. After the ESR, previous glucocorticoid usage, current treatment with nonsteroidal anti-inflammatory drugs, duration of disease and HOMAIR were controlled for, the differences in DHEAS levels between patients and controls were markedly attenuated in women (P = 0.050) and were no longer present in men (P = 0.133). We concluded that low DHEAS concentrations are commonly encountered in IA and, in women, this may not be fully explainable by disease-related parameters. The role of hypoadrenalism in the pathophysiology of IA deserves further elucidation. DHEA replacement may be indicated in many patients with IA, even in those not taking glucocorticoids.

Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone

The structures of the ligand-binding domains (LBD) of the wild-type androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 Å resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wild-type receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor

Whereas several apoptosis-related proteins have been linked to the antiapoptotic effects of Akt serine–threonine kinase, the search continues to explain the Akt signaling role in promoting cell survival via antiapoptotic effects. Here, we demonstrate that Akt phosphorylates the androgen receptor (AR) at Ser-210 and Ser-790. A mutation at AR Ser-210 results in the reversal of Akt-mediated suppression of AR transactivation. Activation of the phosphatidylinositol-3-OH kinase/Akt pathway results in the suppression of AR target genes, such as p21, and the decrease of androgen/AR-mediated apoptosis, which may involve the inhibition of interaction between AR and AR coregulators. Together, these findings provide a molecular basis for cross-talk between two signaling pathways at the level of Akt and AR–AR coregulators that may help us to better understand the roles of Akt in the androgen/AR-mediated apoptosis.

Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. Mutations or abnormal expression of AR in prostate cancer can play a key role in the process that changes prostate cancer from androgen-dependent to an androgen-independent stage. Using a yeast two-hybrid system, we were able to isolate a ligand-dependent AR-associated protein (ARA70), which functions as an activator to enhance AR transcriptional activity 10-fold in the presence of 10(-10) M dihydrotestosterone or 10(-9) M testosterone, but not 10(-6) M hydroxyflutamide in human prostate cancer DU145 cells. Our data further indicated that ARA70 Will only slightly induce the transcriptional activity of other steroid receptors such as estrogen receptor, glucocorticoid receptor, and progesterone receptor in DU145 cells. Together, these data suggest that AR may need a specific coactivator(s) such as ARA70 for optimal androgen activity.

Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.

Androgen responsiveness to competition in humans: the role of cognitive variables

Although androgens are commonly seen as male sex hormones, it has been established over the years that in both sexes, androgens also respond to social challenges. To explain the socially driven changes in androgens, two theoretical models have been proposed: the biosocial model and the challenge hypothesis. These models are typically seen as partly overlapping; however, they generate different predictions that are clarified here. In humans, sports competition and nonmetabolic competitive tasks have been used in the laboratory setting, as a proxy for agonistic interactions in animals. The results reviewed here show that the testosterone (T) response to competition in humans is highly variable – the studies present postcompetition T levels and changes in T that depend on the contest outcome and that cannot be predicted by the current theoretical models. These conflicting results bring to the foreground the importance of considering cognitive factors that could moderate the androgen response to competition. Among these variables, we elect cognitive appraisal and its components as a key candidate modulating factor. It is known that T also modulates the cognitive processes that are relevant to performance in competition. In this article, we reviewed the evidence arising from studies investigating the effect of administering exogenous T and compare those results with the findings from studies that measured endogenous T levels. Finally, we summarized the importance of also considering the interaction between androgens and other hormones, such as cortisol, when investigating the social modulation of T, as proposed by the dual-hormone hypothesis.

Acceptability of short term neo-adjuvant androgen deprivation in patients with locally advanced prostate cancer

Purpose: To determine the acceptability of short term neo-adjuvant maximal androgen deprivation (MAD) to patients treated with external beam radiation for locally advanced prostate cancer. Methods: Between 1996 and 2000, 818 patients with locally advanced, but non-metastatic, prostate cancer were entered into a randomised clinical trial (TROG 96.01), which compared radiation treatment alone with the same radiation treatment and 3 or 6 months neo-adjuvant MAD with goserelin and flutamide. Relevant symptoms, and how troublesome they were to the patient, were scored using a self-assessment questionnaire. This was completed by the patient at registration, and at specified times during and after treatment. Patients taking flutamide had liver function tests checked at regular intervals. Results: All patients have completed at least 12 months follow-up after treatment. Nearly all patients completed planned treatment with goserelin, but 27% of patients in the 6-month MAD treatment arm, and 20% in the 3-month arm, had to stop flutamide early. This was mainly due to altered liver function (up to 17% patients) and bowel side effects (up to 8% patients). However, although flutamide resulted in more bowel symptoms for patients on MAD, there was significant reduction in some urinary symptoms on this treatment. Acute bowel and urinary side effects at the end of radiation treatment were similar in all treatment arms. Side effect severity was unrelated to radiation target volume size, which was reduced by MAD, but symptomatology prior to any treatment was a powerful predictor. Of the 36% of patients who were sexually active before any treatment, the majority became inactive whilst on MAD. However, sexual activity at 12 months after radiation treatment was similar in all treatment arms, indicating that the effects of short term MAD on sexual function are reversible. Conclusion: Despite temporary effects on sexual activity, and compliance difficulties with flutamide, short-term neo-adjuvant MAD was not perceived by patients to be a major inconvenience. If neo-adjuvant MAD in the way tested can be demonstrated to lead to improved biochemical control and/or survival, then patients would view these therapeutic gains as worthwhile. Compliance with short-term goserelin was excellent, confirming that LH-RH analogues have a potential role in more long-term adjuvant treatment. However, for more protracted androgen deprivation, survival advantages and deleterious effects need to be assessed in parallel, in order to determine the optimal duration of treatment. (C) 2003 Elsevier Ireland Ltd. All fights reserved.

Gender diagnosticity and androgen receptor gene CAG repeat sequence

The gender diagnosticity (GD) approach of Lippa (1995) was used to evaluate the relationship of within-sex differences in psychological masculinity-femininity to a genetic characteristic, the length of a repeated CAG sequence in the X-linked androgen receptor (AR) gene. Previously assessed adult samples in Australia and Sweden were used for this purpose. A weak relationship (correlations in the range .11 to .14) was obtained in both countries. Additional data from adolescent twins from Australia (12-, 14-, 16-year-olds) did not confirm such a relationship at those ages, especially for males. The fact that this sample consisted of twins permitted two kinds of within-pair comparisons: (1) Did the dizygotic twin who had the longer AR sequence have the higher GD score? (2) Was one twin's GD score more highly correlated with the other twin's AR score in MZ than in DZ pairs? The answer in both cases was negative. Clarification of these relationships will require large samples and measurements at additional ages.

Polymorphic CAG repeat length in the androgen receptor gene and association with neurodegeneration in a heterozygous female carrier of Kennedy's disease

Kennedy's disease (spinobulbar muscular atrophy) is an X-linked form of motor neuron disease affecting adult males carrying a CAG trinucleotide repeat expansion within the androgen receptor gene. While expression of Kennedy's disease is thought to be confined to males carrying the causative mutation, subclinical manifestations have been reported in a few female carriers of the disease. The reasons that females are protected from the disease are not clear, especially given that all other diseases caused by CAG expansions display dominant expression. In the current study, we report the identification of a heterozygote female carrying the Kennedy's disease mutation who was clinically diagnosed with motor neuron disease. We describe analysis of CAG repeat number in this individual as well as 33 relatives within the pedigree, including two male carriers of the Kennedy's mutation. The female heterozygote carried one expanded allele of the androgen receptor gene with CAG repeats numbering in the Kennedy's disease range (44 CAGs), with the normal allele numbering in the upper-normal range (28 CAGs). The subject has two sons, one of whom carries the mutant allele of the gene and has been clinically diagnosed with Kennedy's disease, whilst the other son carries the second allele of the gene with CAGs numbering in the upper normal range and displays a normal phenotype. This coexistence of motor neuron disease and the presence of one expanded allele and one allele at the upper limit of the normal range may be a coincidence. However, we hypothesize that the expression of the Kennedy's disease mutation combined with a second allele with a large but normal CAG repeat sequence may have contributed to the motor neuron degeneration displayed in the heterozygote female and discuss the possible reasons for phenotypic expression in particular individuals.