Coordinated expression of oestrogen and androgen receptors in HER2-positive breast carcinomas: impact on proliferative activity

Aims Human epidermal growth factor receptor 2 (HER2)-positive breast cancers are aggressive neoplasms associated with a variable response to systemic therapies. Therefore, the identification of biomarkers to better characterise this heterogeneity would improve treatment efficacy. The aim of this study was to evaluate the influence of androgen receptor (AR) and oestrogen receptor (ER) on clinicopathological features in a series of HER2-positive breast carcinomas. Methods A total of 104 carcinomas were selected and reviewed. Immunohistochemical studies for ER, progesterone receptor and Ki-67 were analysed on tumour whole histological sections. AR expression was analysed on samples represented on tissue microarrays. According to steroid receptor expression, cases were classified into three groups: AR positive/ER positive (48 cases), AR positive/ER negative (41 cases) and AR negative/ER negative (13 cases). Results AR-positive tumours corresponded to 89 (85.6%) of 104 carcinomas. AR-positive carcinomas were associated with a higher frequency of ER and progesterone receptor co-expression and lower proliferative activity determined by the expression of Ki-67. AR-negative carcinomas were more often high grade. The group of AR-positive/ER-negative carcinomas was associated with the highest frequency of apocrine morphological features. The group of AR-negative/ER-negative carcinomas was associated with the highest proliferative activity and the highest frequency of high histological and nuclear grade. The lowest frequency of high-grade tumours and the lowest proliferative activity were seen among tumours with expression of both receptors. Conclusions These results suggest that co-expression of AR and ER can provide a protective effect based on phenotypical presentation of HER2-positive carcinomas. Furthermore, lack of both steroid hormone receptors characterises the most aggressive phenotype.

Androgen receptors are differentially expressed in Gleason patterns of prostate cancer and down-regulated in matched lymph node metastases

Androgen receptor (AR) expression profile in the different Gleason patterns (GP) of primary prostate cancers and nodal metastases is unknown. More information about AR distribution is needed to optimize evaluation methods and to better understand the role of AR in development and progression of prostate cancer.

A novel steroidal antiandrogen targeting wild type and mutant androgen receptors

Prostate cancer (PCa) progression is enhanced by androgen and treatment with antiandrogens represents an alternative to castration. While patients initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years by expressing androgen receptor (AR) mutants. Such mutations, indeed, promote unfavorable agonistic behavior from classical antagonists. Here, we have synthesized and screened 37 novel compounds derived from dihydrotestosterone (DHT), cyanolutamide and hydroxyflutamide. These derivatives were tested for their potential antagonistic activity using a luciferase reporter gene assay and binding properties were determined for wild type (WT) and mutant ARs (T877A, W741C, W741L, H874Y). In the absence and presence of antiandrogens, androgen dependent cellular proliferation and prostate specific antigen (PSA) expression were assayed in the prostate cancer cell line LNCaP by crystal violet, real time PCR and by Western blots. Also, cellular proliferation and PSA expression were assayed in 22Rv1. A novel compound RB346, derived from DHT, was found to be an antagonist for all tested AR forms, preventing DHT induced proliferation and PSA expression in LNCaP and 22Rv1 cells. RB346 displayed no agonistic activity, in contrast to the non-steroidal antiandrogen bicalutamide (Casodex) with unfavorable agonistic activity for W741L-AR. Additionally, RB346 has a slightly higher binding affinity for WT-AR, T877A-AR and H874Y-AR than bicalutamide. Thus, RB346 is the first potent steroidal antiandrogen with efficacy for WT and various AR mutants.

Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca2+], which is due to release of Ca2+ from intracellular Ca2+ stores. This Ca2+ mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

Estrogen receptor-Beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNF-alpha mediated

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor–β (ERβ) in BPH and PCa. ERβ agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFα knock-out mice fail to respond to ERβ agonist, demonstrating the requirement for TNFα signaling. In human tissues, ERβ agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133+ enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERβ causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERβ agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERβ agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

Detection of mRNA levels for the estrogen alpha, estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue

Previous studies in our laboratory have shown association of nuclear receptor expression and histological breast cancer grade. To further investigate these findings, it was the objective of this study to determine if expression levels of the estrogen alpha, estrogen beta and androgen nuclear receptor genes varied in different breast cancer grades. RNA extracted from paraffin embedded archival breast tumour tissue was converted into cDNA and cDNA underwent PCR to enable quantitation of mRNA expression. Expression data was normalised against the 18S ribosomal gene multiplex and analysed using ANOVA. Analysis indicated a significant alteration of expression for the androgen receptor in different cancer grades (P=0.014), as well as in tissues that no longer possess estrogen receptor alpha proteins (P=0.025). However, expression of estrogen receptors alpha and beta did not vary significantly with cancer grade (P=0.057 and 0.622, respectively). Also, the expression of estrogen receptor alpha or beta did not change, regardless of the presence of estrogen receptor alpha protein in the tissue (P=0.794 and 0.716, respectively). Post-hoc tests indicate that the expression of the androgen receptor is increased in estrogen receptor negative tissue as well as in grade 2 and grade 3 tumours, compared to control tissue. This increased expression in late stage breast tumours may have implications to the treatment of breast tumours, particularly those lacking expression of other nuclear receptor genes.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Bone morphogenetic proteins (BMP)-4,-6, and-7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: Could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and - 7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, - 6, and - 7 potently suppress both basal ( P < 0.0001; respective IC50 values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced ( P < 0.0001; respective IC50 values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3 beta-hydroxysteroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment ( P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and - 7, respectively, and the observation that both antagonists enhanced ( P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP- 4 and - 7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.

Theca cells and the regulation of ovarian androgen production

Theca cells are essential for female reproduction being the source of androgens that are precursors for follicular oestrogen synthesis and also signal through androgen receptors (AR) in the ovary and elsewhere. Theca cells arise from mesenchymal cells around the secondary follicle stage. Their recruitment, proliferation and cytodifferentiation are influenced, directly or indirectly, by paracrine signals from granulosa cells and oocyte although uncertainty remains over which are the critically important signals at particular stages. In a reciprocal manner, theca cells secrete factors that influence granulosa cell proliferation and differentiation at different follicle stages. Differentiated theca interna cells acquire responsiveness to luteinizing hormone (LH) and other endocrine signals and express components of the steroidogenic machinery required for androgen biosynthesis. They also express insulin-like peptide 3 (INSL3) and its receptor (RXFP2), levels of which increase during bovine antral follicle development. INSL3 signaling may play a role in promoting androgen biosynthesis since knockdown of either INSL3 or its receptor (RXFP2) in bovine theca cells inhibits androgen biosynthesis while exogenous INSL3 can raise androgen secretion. Bone morphogenetic proteins (BMPs) of thecal or granulosal origin suppress thecal production of both INSL3 and androgen. Inhibin, produced in greatest amounts by granulosa cells of preovulatory follicles, reverses these BMP actions. Thus, BMP-induced inhibition of thecal androgen production may be mediated by reduced INSL3-RXFP2 signaling. Activins also inhibit androgen production in an inhibin-reversible manner and recent evidence in sheep indicates that theca cells synthesize and secrete activin, implying an autocrine role in suppressing androgen biosynthesis in smaller follicles, akin to that envisaged for BMPs.

Physical exercise on the rat ventral prostate: Steroid hormone receptors, apoptosis and cell proliferation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of combined hormonal and insulin therapy on the steroid hormone receptors and growth factors signalling in diabetic mice prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Androgen receptor in the Mongolian gerbil ventral prostate: Evaluation during different phases of postnatal development and following androgen blockage

The normal growth, differentiation and maintenance of the morphofunctional integrity of the prostate gland are dependent on the interaction of constant levels of androgens with their receptors. The need to study the responses to hormones under several conditions and the effect of their blockage is due to the fact that the human prostate is the site of a great number of age-related diseases, and the ones with a major medical importance are prostate cancer (Cap) and benign prostatic hyperplasia (BPH), which can both be treated with androgen suppression. Seventy-five male gerbils were divided, randomly, into 3 groups of 25 animals each, where each group corresponded to one phase of postnatal development. In each phase, it was possible to morphologically and stereologically analyze the compartments of prostatic ventral lobe, as well as to immunohistochemically analyze the degree of expression of androgen receptors (ARs) after the androgen blockage therapies. In addition, it was possible to establish the hormonal dosage of serum testosterone levels given the comparative approach of the expression of androgen receptors. There is a pattern of AR distribution in the prostatic ventral lobe throughout postnatal development, in which the younger the animal is the higher, the interaction of circulating androgens that stimulate the AR expression in both the epithelial and stromal compartments. The androgen blockage therapies decreased AR expression in the prostatic compartments, but the androgen reposition after these blockages was not sufficient to recover the glandular structure or stimulate the AR expression up to normal physiological conditions. Both the regulation and distribution of androgen receptors along the gerbil prostatic tissues are complex mechanisms that are likely to be genetically regulated by androgens prenatally or by other factors that are still unknown. This rodent species seems to be a valuable model in the attempt to improve the understanding of the morphophysiological and pathological behavior of this important gland in humans throughout aging and to stimulate new therapeutic ideas to fight prostate cancer. (C) 2008 Elsevier Ltd. All rights reserved.