Phenotypic and metabolic aspects of prostatic epithelial cells in aged gerbils after antisteroidal therapy: Turnover in the state of chromatin condensation and androgen-independent cell replacement

The gerbil is a rodent considered a good model for studies of prostatic morphophysiology under different experimental conditions. Studies involving castration and steroidal blockers of aged gerbils showed that the glandular epithelium persists after long-term therapy, preventing the organ atrophy. Thus, the objective of this study was to evaluate the phenotypic characteristics and behavior of prostatic epithelial cells that remained after different periods of hormone ablation in aged gerbils. The identification of elements that influenced the survival of this cell type was performed by morphometric, nuclear phenotypes, ultrastructural and immune histochemical analysis. The most significant responses to treatment, by analyzing morphometric features, were observed during the first three time points (day 1, day 3, and day 7), after which there appeared to be an adjustment of the gland to the hormone ablation. All treatments led to changes in the state of chromatin condensation, DNA methylation pattern and phenotypic changes indicated cell senescence. Additionally, an increase in the basal cells seemed to guarantee self-renewal properties to the epithelium. These data indicate that changes occur at many levels, including gene expression and nuclear architecture in the epithelial cells, when aging and steroidal blockade are associated. These aspects are important when considering castration-resistant prostate cancer, a malignant tumor posing difficult therapeutic intervention. © 2013 Elsevier GmbH. All rights reserved.

Excess androgen during perinatal life alters steroid receptor expression, apoptosis, and cell proliferation in the uteri of the offspring

Exposure to environmental chemicals may contribute to reproductive disorders, especially when it occurs in critical periods of development. The female reproductive system can be a target for androgens derived from environmental contaminants or pathological conditions. The purpose of this study was to assess the long-term effects of androgens on uterine tissue after maternal exposure limited to the time of gestation and lactation. Pregnant Wistar rats were treated with testosterone propionate (TP) at 0.05. mg/kg, 0.1. mg/kg, 0.2. mg/kg or corn oil (vehicle), s.c., from gestational day 12 until the end of lactation. The results show changes in the pattern of expression of receptors for estrogen, progesterone, and androgen at all doses tested, and decreases in both apoptosis and cell proliferation indices at 0.1 and 0.2. mg/kg. We conclude that early TP exposure, under these experimental conditions, causes changes in cellular and molecular parameters that are essential for normal uterine function in the adult. © 2013 Elsevier Inc.

Androgen therapy reverses injuries caused by ethanol consumption in the prostate: testosterone as a possible target to ethanol-related disorders

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

Androgen receptors and experimental bone loss - an in vivo and in vitro study

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

Perinatal exposure to androgen excess and the effects on the rat uterine estradiol responsiveness

Androgen exposure during sexual development induces alterations in steroidal target tissues. The objective of this study was to evaluate the uterine responsiveness to estradiol after perinatal androgenization. Pregnant Wistar rats were exposed to corn oil or testosterone propionate at 0.05, 0.1, or 0.2 mg/kg from gestational day 12 until postnatal day 21. Female offspring was challenged with estradiol (E2 ) after weaning (0.4 mg/kg) and at adulthood (10 or 100 µg/day), when the pituitary response was also evaluated. At adulthood, control and 0.05 mg/kg groups presented a uterine weight increment when exposed to 100 µg/day of E2 , 0.1 mg/kg group only responded to 10 µg/day of E2 , and the 0.2 mg/kg group showed increased uterine weight at both doses. The pituitary weight was similarly increased after estradiol stimulation in all experimental groups. In conclusion, testosterone propionate exposure induced an abnormal stimulation of uterine tissue growth by estrogen stimulus without affecting pituitary response. More studies are needed to clarify whether these alterations are capable of impairing the reproductive capacity of the female tract. © 2015 Wiley Periodicals, Inc. Environ Toxicol, 2015.

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.

Surrogate endpoints for prostate cancer-specific mortality after radiotherapy and androgen suppression therapy in men with localised or locally advanced prostate cancer: an analysis of two randomised trials

Background Androgen suppression therapy and radiotherapy are used to treat locally advanced prostate cancer. 3 years of androgen suppression confers a small survival benefit compared with 6 months of therapy in this setting, but is associated with more toxic effects. Early identification of men in whom radiotherapy and 6 months of androgen suppression is insufficient for cure is important. Thus, we assessed whether prostate-specific antigen (PSA) values can act as an early surrogate for prostate cancer-specific mortality (PCSM). Methods We systematically reviewed randomised controlled trials that showed improved overall and prostate cancer-specific survival with radiotherapy and 6 months of androgen suppression compared with radio therapy alone and measured lowest PSA concentrations (PSA nadir) and those immediately after treatment (PSA end). We assessed a cohort of 734 men with localised or locally advanced prostate cancer from two eligible trials in the USA and Australasia that randomly allocated participants between Feb 2, 1996, and Dec 27, 2001. We used Prentice criteria to assess whether reported PSA nadir or PSA end concentrations of more than 0.5 ng/mL were surrogates for PCSM. Findings Men treated with radiotherapy and 6 months of androgen suppression in both trials were significantly less likely to have PSA end and PSA nadir values of more than 0.5 ng/mL than were those treated with radiotherapy alone (p<0.0001). Presence of candidate surrogates (ie, PSA end and PSA nadir values >0.5 ng/mL) alone and when assessed in conjunction with the randomised treatment group increased risk of PCSM in the US trial (PSA nadir p=0.0016; PSA end p=0.017) and Australasian trial (PSA nadir p<0.0001; PSA end p=0.0012). In both trials, the randomised treatment group was no longer associated with PCSM (p >= 0.20) when the candidate surrogates were included in the model. Therefore, both PSA metrics satisfied Prentice criteria for surrogacy. Interpretation After radiotherapy and 6 months of androgen suppression, men with PSA end values exceeding 0.5 ng/mL should be considered for long-term androgen suppression and those with localised or locally advanced prostate cancer with PSA nadir values exceeding 0.5 ng/mL should be considered for inclusion in randomised trials investigating the use of drugs that have extended survival in castration-resistant metastatic prostate cancer.

Androgen responsive intronic non-coding RNAs

Abstract Background Transcription of large numbers of non-coding RNAs originating from intronic regions of human genes has been recently reported, but mechanisms governing their biosynthesis and biological functions are largely unknown. In this work, we evaluated the existence of a common mechanism of transcription regulation shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line. Results Using a custom-built cDNA microarray enriched in intronic transcribed sequences, we found 39 intronic non-coding RNAs for which levels were significantly regulated by androgen exposure. Orientation-specific reverse transcription-PCR indicated that 10 of the 13 were transcribed in the antisense direction. These transcripts are long (0.5–5 kb), unspliced and apparently do not code for proteins. Interestingly, we found that the relative levels of androgen-regulated intronic transcripts could be correlated with the levels of the corresponding protein-coding gene (asGAS6 and asDNAJC3) or with the alternative usage of exons (asKDELR2 and asITGA6) in the corresponding protein-coding transcripts. Binding of the androgen receptor to a putative regulatory region upstream from asMYO5A, an androgen-regulated antisense intronic transcript, was confirmed by chromatin immunoprecipitation. Conclusion Altogether, these results indicate that at least a fraction of naturally transcribed intronic non-coding RNAs may be regulated by common physiological signals such as hormones, and further corroborate the notion that the intronic complement of the transcriptome play functional roles in the human gene-expression program.

Androgen receptor gene polymorphisms lean mass and performance in young men

[EN] The exon-1 of the androgen receptor (AR) gene contains two repeat length polymorphisms which modify either the amount of AR protein inside the cell (GGN(n), polyglycine) or its transcriptional activity (CAG(n), polyglutamine). Shorter CAG and/or GGN repeats provide stronger androgen signalling and vice versa. To test the hypothesis that CAG and GGN repeat AR polymorphisms affect muscle mass and various variables of muscular strength phenotype traits, the length of CAG and GGN repeats was determined by PCR and fragment analysis and confirmed by DNA sequencing of selected samples in 282 men (28.6 +/- 7.6 years). Individuals were grouped as CAG short (CAG(S)) if harbouring repeat lengths of 21. GGN was considered short (GGN(S)) or long (GGN(L)) if GGN 23, respectively. No significant differences in lean body mass or fitness were observed between the CAG(S) and CAG(L) groups, or between GGN(S) and GGN(L) groups, but a trend for a correlation was found for the GGN repeat and lean mass of the extremities (r=-0.11, p=0.06). In summary, the lengths of CAG and GGN repeat of the AR gene do not appear to influence lean mass or fitness in young men.

Bone mass and the CAG and GGN androgen receptor polymorphisms in young men

[EN] BACKGROUND: To determine whether androgen receptor (AR) CAG (polyglutamine) and GGN (polyglycine) polymorphisms influence bone mineral density (BMD), osteocalcin and free serum testosterone concentration in young men. METHODOLOGY/PRINCIPAL FINDINGS: Whole body, lumbar spine and femoral bone mineral content (BMC) and BMD, Dual X-ray Absorptiometry (DXA), AR repeat polymorphisms (PCR), osteocalcin and free testosterone (ELISA) were determined in 282 healthy men (28.6+/-7.6 years). Individuals were grouped as CAG short (CAG(S)) if harboring repeat lengths of < or = 21 or CAG long (CAG(L)) if CAG > 21, and GGN was considered short (GGN(S)) or long (GGN(L)) if GGN < or = 23 or > 23. There was an inverse association between logarithm of CAG and GGN length and Ward's Triangle BMC (r = -0.15 and -0.15, P<0.05, age and height adjusted). No associations between CAG or GGN repeat length and regional BMC or BMD were observed after adjusting for age. Whole body and regional BMC and BMD values were similar in men harboring CAG(S), CAG(L), GGN(S) or GGN(L) AR repeat polymorphisms. Men harboring the combination CAG(L)+GGN(L) had 6.3 and 4.4% higher lumbar spine BMC and BMD than men with the haplotype CAG(S)+GGN(S) (both P<0.05). Femoral neck BMD was 4.8% higher in the CAG(S)+GGN(S) compared with the CAG(L)+GGN(S) men (P<0.05). CAG(S), CAG(L), GGN(S), GGN(L) men had similar osteocalcin concentration as well as the four CAG-GGN haplotypes studied. CONCLUSION: AR polymorphisms have an influence on BMC and BMD in healthy adult humans, which cannot be explained through effects in osteoblastic activity.

Involvement of microRNAs in Androgen Receptor-dependent Breast Cancers

Triple negative breast cancer (TNBC) is a very aggressive tumor subtype characterized by the lack of expression of estrogen receptor 1 (ESR1), due in the most of cases to an increased expression of DNA methyltransferases (DNMTs) and hypermethylation in CpG islands, resulting in gene silencing. Furthermore, in ESR1- negative breast cancers, androgen receptor (AR) is highly expressed and some studies suggest that it can drive tumor progression and might represent a therapeutic target. A correlation between microRNAs, small non-coding RNAs that regulate gene expression, and DNMTs was investigated in a TNBC cell line to restore a normal methylation pattern of ESR1, leading to its re-expression and conferring again sensitivity to selective estrogen receptor modulators (SERMs). miR-148A and miR-29B were found to be involved in the reduction of the expression of DNMT1 and DNMT3A and in a slight increase of ESR1 expression, but not at protein level. Then, we found a down-regulation of AR by miRs-7, -9, -27a, -27b, -29a, -29b, -29c, -127-3p, -127-5p and -376 at 48h post transfection and an up-regulation by miR-15a and miR-16 at every time considered. We concomitantly investigated a possible increase of Tamoxifen, Herceptin and Metformin sensitivity after AR silencing in MDA-MB 453 and T-47D cell lines. Cells seemed more sensitive when silenced for AR only in MDA-MB-453 at 24h post Tamoxifen treatment. Studies on Metformin have basically confirmed an increase of drug sensitivity due to AR silencing in both cell lines. Analysis of Herceptin showed how MDA-MB 453 samples silenced for AR have a slight decrease in the percentage of proliferating cells, demonstrating a possible increase in the response to treatment. These preliminary data provide the basis for further study of the modulation of the expression of AR by microRNAs and it will be interesting to understand the molecular mechanisms underlying these interactions.

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.

Impact of differential P450c17 phosphorylation by cAMP stimulation and by starvation conditions on enzyme activities and androgen production in NCI-H295R cells

CYP17A1 plays a pivotal role in the biosynthesis of androgens in the adrenals and the gonads. Although this enzyme catalyzes two different reactions on one single active site, its specific activities are regulated independently. Although the 17alpha-hydroxylase activity is rather constant and regulated by gene expression, the 17,20-lyase activity varies significantly with the amount of cofactors or by protein phosphorylation. cAMP increases CYP17A1 expression, P450c17 phosphorylation, and androgen production. However, the exact mechanism(s) and the specific regulators of CYP17A1 remain unknown. Therefore, we studied the regulation of adrenal androgen biosynthesis in human adrenal H295R cells focusing on CYP17A1. We analyzed androgen production and P450c17 activities in H295R cells grown under normal and serum-free conditions and/or after stimulation with 8-bromoadenosine-cAMP. H295R cells grown in starvation medium produced more androgens and had decreased HSD3B2 expression and activity but increased P450c17-17,20-lyase activity and serine phosphorylation. Although starvation increased serine phosphorylation of P450c17 specifically, cAMP stimulation enhanced threonine phosphorylation exclusively. Time-course experiments revealed that a short cAMP stimulation augmented threonine phosphorylation of P450c17 but did not increase 17,20-lyase activity. By contrast, long cAMP stimulation increased androgen production through increased P450c17 activities by enhancing CYP17A1 gene expression. We conclude that serum withdrawal shifts steroidogenesis of H295R cells towards androgen production, providing a suitable model for detailed studies of androgen regulation. In addition, our study shows that starvation and cAMP stimulation regulate P450c17 phosphorylation differentially and that an increase in P450c17 phosphorylation does not necessarily lead to enhanced enzyme activity and androgen production.