mibolerone and Breast-Neoplasms

Although the protective role of androgen receptor (AR) in breast cancer (BC) is well established, the mechanisms involved remains largely unexplored. MicroRNAs play fundamental roles in many biological processes, including tumor cell development and metastasis. Herein, we report that androgens reduce BC cells proliferation acting as a negative modulator of the onco-miRNA-21.The synthetic androgen miboleron (Mib) decreases BC cell proliferation induced by miR-21 over-expression and AR knockdown evidenced the requirement of AR in the down-regulation of miR-21 expression. These effects seem to be a general mechanism occurring in BC tissues.Chromatin immune-precipitation (ChIP) analysis disclosed the binding of AR to a specific ARE sequence in miR-21 proximal promoter and recognizes the recruitment of HDAC3 as component for AR-mediated transcriptional repression. Such event is associated to a significantly reduced PolII binding in Mib treated extracts confirming that activated AR is a transcriptional repressor of miR-21 expression, providing further insight into the protective role of androgens in breast cancer cells.Collectively, our data and the widespread AR expression in primary and metastatic breast tumours, suggest a careful examination of the therapeutic potential of androgens also in potentiating the effectiveness of anti-oestrogen adjuvant therapies.

Topics: Androgens; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Nandrolone; Receptors, Androgen

The two isoforms of estrogen receptor (ER) alpha and beta play opposite roles in regulating proliferation and differentiation of breast cancers, with ER-alpha mediating mitogenic effects and ER-beta acting as a tumor suppressor. Emerging data have reported that androgen receptor (AR) activation inhibits ER-positive breast cancer progression mainly by antagonizing ER-alpha signaling. However, to date no studies have specifically evaluated a potential involvement of ER-beta in the inhibitory effects of androgens.. ER-beta expression was examined in human breast cancer cell lines using real-time PCR, Western blotting and small interfering RNA (siRNA) assays. Mutagenesis studies, electromobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were performed to assess the effects of mibolerone/AR on ER-beta promoter activity and binding.. In this study, we demonstrate that mibolerone, a synthetic androgen ligand, up-regulates ER-beta mRNA and protein levels in ER-positive breast cancer cells. Transient transfection experiments, using a vector containing the human ER-beta promoter region, show that mibolerone increases basal ER-beta promoter activity. Site-directed mutagenesis and deletion analysis reveal that an androgen response element (ARE), TGTTCT motif located at positions -383 and -377, is critical for mibolerone-induced ER-beta up-regulation in breast cancer cells. This occurs through an increased recruitment of AR to the ARE site within the ER-beta promoter region, along with an enhanced occupancy of RNA polymerase II. Finally, silencing of ER-beta gene expression by RNA interference is able to partially reverse the effects of mibolerone on cell proliferation, p21 and cyclin D1 expression.. Collectively, these data provide evidence for a novel mechanism by which activated AR, through an up-regulation of ER-beta gene expression, inhibits breast cancer cell growth.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Activation; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; MCF-7 Cells; Mutagenesis, Site-Directed; Nandrolone; Promoter Regions, Genetic; Protein Binding; Receptors, Androgen; rho GTP-Binding Proteins; RNA Interference; RNA Polymerase II; RNA, Messenger; RNA, Small Interfering; Testosterone Congeners; Up-Regulation

Sexual hormones, estrogens and androgens, determine biological response in a tissue- and gender-specific manner and have a pivotal role in endocrine-mediated tumorigenesis. In situ estrogen production by aromatase is a critical determinant for breast cancer growth and progression. On the contrary, clinical and in vitro studies indicate that androgens have a protective role in mammary carcinogenesis. Here, we demonstrated, in hormone-dependent breast cancer cells, the existence of a functional interplay between the androgen receptor (AR), the orphan nuclear receptor DAX-1 and the aromatase enzyme involved in the inhibition of the estrogen-dependent breast cancer cell proliferation exerted by androgen signaling. Indeed, our results revealed, in MCF-7 cells, that ligand-activated AR induces the expression of the orphan nuclear receptor DAX-1 by direct binding to a newly identified androgen-response-element within the DAX-1 proximal promoter. In turn, androgen-induced DAX-1 is recruited, in association with the corepressor N-CoR, within the SF-1/LRH-1 containing region of the aromatase promoter, thereby repressing aromatase expression and activity. In elucidating a novel mechanism by which androgens, through DAX-1, inhibit aromatase expression in breast cancer cell lines, these findings reinforce the theory of androgen- opposing estrogen-action, opening new avenues for therapeutic intervention in estrogen-dependent breast tumors.

Topics: Androgens; Apoptosis; Aromatase; Base Sequence; Breast Neoplasms; Cell Proliferation; DAX-1 Orphan Nuclear Receptor; Enzyme Repression; Estrogens; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Nandrolone; Neoplasms, Hormone-Dependent; Promoter Regions, Genetic; Receptors, Androgen; Response Elements

Androgen signaling, mediated by the androgen receptor (AR), is a critical factor influencing growth of normal and malignant breast cells. Given the increasing use of exogenous androgens in women, a better understanding of androgen action in the breast is essential. This study compared the effects of 5alpha-dihydrotestosterone (DHT) and a synthetic androgen, mibolerone, on estradiol (E(2))-induced proliferation of breast cancer cells. DHT modestly inhibited E(2)-induced proliferation and mibolerone significantly inhibited proliferation in T-47D cells. The effects of both androgens could be reversed by an AR antagonist, suggesting that their actions were mediated, in part, by AR. Whereas high physiological doses (10-100nM) of DHT reduced E(2)-mediated induction of the estrogen-regulated gene progesterone receptor (PR) to basal levels, mibolerone at lower doses (1nM) eliminated PR expression, suggesting that mibolerone may also act via the PR. In the AR positive, PR-negative MCF-7 cells, mibolerone had modest effects on E(2)-induced proliferation, but was a potent inhibitor of proliferation in the AR positive, PR positive MCF-7M11 PRA cells. The effects of mibolerone in breast cancer cells were similar to those of the progestin, medroxyprogesterone acetate. Our results demonstrate that mibolerone can have both androgenic and progestagenic actions in breast cancer cells.

Topics: Androgens; Breast Neoplasms; Cell Proliferation; Dihydrotestosterone; Drug Evaluation, Preclinical; Estradiol; Humans; Medroxyprogesterone Acetate; Nandrolone; Progestins; Receptors, Androgen; Receptors, Progesterone; Testosterone Congeners; Tumor Cells, Cultured

We have developed a competitive assay to measure the estrogen-regulated protein pS2. A monoclonal pS2 antibody (mAb) and a biotinylated pS2 peptide are used, with time-resolved fluorometry as a detection technique. The assay has a detection limit of 16 ng/mL and is precise (within-run and day-to-day Cvs 3-12%). We used this assay to determine steroid hormone activity of six steroids in cell culture, both in terms of time course and dose response. pS2 concentrations in the tissue culture supernatant of the BT-474 breast carcinoma cell line were significantly higher when estradiol was the stimulating steroid. There was a significant time course and dose response observed for estradiol, but not for the other steroids. The availability of a sensitive, reliable, and convenient method for quantifying pS2 will allow for many research applications including the screening of natural and synthetic compounds for putative estrogenic activity.

Topics: Aldosterone; Amino Acid Sequence; Antibodies, Monoclonal; Binding, Competitive; Biotin; Breast Neoplasms; Calibration; Chemistry, Clinical; Dexamethasone; Dihydrotestosterone; Dose-Response Relationship, Immunologic; Drosophila Proteins; Estradiol; Evaluation Studies as Topic; Fluorescent Antibody Technique; Fluorometry; Glucocorticoids; Integrin alpha Chains; Integrins; Molecular Sequence Data; Nandrolone; Norgestrel; Progesterone Congeners; Sensitivity and Specificity; Testosterone Congeners; Tumor Cells, Cultured

Sex steroids control the proliferation of their target cells through two different pathways: 1) proliferative response (Step-1); and 2) inhibition of cell proliferation (Step-2). Mechanisms of cell proliferation regulation are incompletely understood; however, there is general agreement with the notion that sex steroid receptors play an important role in the control of the proliferation of sex steroid target cells. To test this hypothesis, a full human androgen receptor (AR) vector was transfected into human breast cancer MCF7 cells. The cloned cells that stably express the AR, called MCF7-AR1 cells, contained approximately five times more AR than the wild-type MCF7 cells from which they were derived. These AR-transfected cells retained their capacity to proliferate when estrogens were added to 10% charcoal-dextran stripped human serum but did not acquire the ability to proliferate when androgens were added to this medium. In serumless medium (ITDME), these cells proliferated maximally, as MCF7 cells did; however, natural and synthetic androgens prevented the AR-transfected cells from proliferating. Inhibition of cell proliferation occurred when physiological androgen concentrations (1 nM) were added to ITDME; this effect was almost completely reversed by Casodex, a synthetic androgen antagonist. Under the effect of androgens added to ITDME, MCF7-AR1 cells were arrested in the G0/G1 phase within 24 h. These data suggest that: 1) the androgen-induced inhibition of cell proliferation (Step-2) is AR-mediated; and 2) the AR may be necessary, but not sufficient, to mediate the androgen-induced proliferative response (Step-1).

Topics: Androgen Antagonists; Androgens; Anilides; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Division; Dihydrotestosterone; Dose-Response Relationship, Drug; Female; Flutamide; Humans; In Vitro Techniques; Metribolone; Nandrolone; Nitriles; Receptors, Androgen; Testosterone; Testosterone Congeners; Tosyl Compounds; Transfection; Tumor Cells, Cultured

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.

Topics: Androgen Receptor Antagonists; Androgens; Breast Neoplasms; Cell Division; Dihydrotestosterone; Flutamide; Humans; Imidazoles; Imidazolidines; Nandrolone; Oligonucleotides, Antisense; Receptors, Androgen; Tumor Cells, Cultured

Using synthetic peptides with sequences derived from specific regions of human estrogen (ER) and progesterone (PR) receptors, we have developed site-directed monoclonal and polyclonal antibodies to specific domains of these receptors. These antibodies interact specifically with the native (nondenatured) receptors and detect changes in the conformation of these proteins. Monoclonal antibody PR-AT 4.14 bound more tightly to PR-ORG 2058 complexes than to PR-7 alpha,17 alpha-dimethyl-19-nor-testosterone (7 alpha,17 alpha, DMNT; mibolerone) complexes, suggesting possible ligand-induced conformational changes in PR. In the absence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 4S-5S entity on sucrose density gradients (SDG) containing 0.4 M KCl. In the presence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 7-8S complexes. In contrast, at the same concentrations of antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes sedimented at 4S-5S region in the absence of the antibody and as two populations in the presence of the antibody, suggesting that the antibody did not recognize all of the PR-7 alpha,17 alpha, DMNT complexes. To exclude the possibility that the inability of the antibody to recognize receptor-[3H]7 alpha,17 alpha, DMNT complexes was due to its binding to androgen receptors, unlabeled 5 alpha-dihydrotestosterone (5 alpha-DHT) (50 nM) was added to the incubation to inhibit 7 alpha,17 alpha, DMNT binding to androgen receptors. While PR-[3H]ORG 2058 complexes were immunoprecipitated in the presence of the antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes were only partially immunoprecipitated, further confirming the results obtained with SDG.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding Sites, Antibody; Binding, Competitive; Blotting, Western; Breast Neoplasms; Cattle; Female; Humans; Nandrolone; Precipitin Tests; Pregnenediones; Protein Conformation; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Receptors, Progesterone; Uterus

The role of androgens and the androgen receptor (AR) in the development and progression of breast cancer is poorly understood. To further define a potential model for androgen action in breast cancer, MDA-MB-453 cells, which express AR in the absence of oestrogen receptors and progesterone receptors, were further characterised in terms of AR expression and androgen responsiveness. High level expression of AR was confirmed by northern blot analysis, radioligand binding and immunocytochemistry, and could not be accounted for by AR gene amplification. Three endogenous androgen-responsive genes (fatty acid synthetase, gross cystic disease fluid protein of 15 kDa and prolactin receptor) and a transfected reporter gene, containing an androgen-responsive element, were induced following androgen administration. A synthetic androgen, mibolerone, induced moderate (27% above control) stimulation of MDA-MB-453 cell proliferation, which was abrogated by the simultaneous administration of the synthetic androgen antagonist, anandron, demonstrating that the effect was AR-mediated. In summary, MDA-MB-453 cells express high levels of functional AR, and thus provide a valuable in vitro model for further studies on androgen regulation of gene expression, and perhaps cell proliferation in breast cancer.

Topics: Blotting, Southern; Breast Neoplasms; Cell Division; Dihydrotestosterone; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Middle Aged; Nandrolone; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Testosterone Congeners; Tumor Cells, Cultured

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 nM 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 S1 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 transcription 96 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 cells 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 vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Topics: Androgens; Antibodies; Base Sequence; Blotting, Northern; Breast Neoplasms; Cells, Cultured; Down-Regulation; Fibroblasts; Gene Expression Regulation; Humans; Immunohistochemistry; Male; Molecular Sequence Data; Nandrolone; Prostatic Neoplasms; Protein Processing, Post-Translational; Receptors, Androgen; RNA, Messenger; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured