alpha-tocopherol affects androgen metabolism in male rat

The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study has provided the first evidence implicating vitamin E in hormone synthesis. The effect of vitamin E on stereoidogenesis in testes and adrenal glands was assessed in growing rats using Affymetrix gene-chip technology. Dietary supplementation of rats with vitamin E (60 mg/kg feed) for a period of 429 days caused a significant repression of genes encoding for proteins centrally involved in the uptake (low-density lipoprotein receptor) and de novo synthesis (for example, 7-dehydrocholesterol reductase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, isopentenyl-diphosphate delta-isomerase, and farnesyl pyrophosphate synthetase) of cholesterol, the precursor of all steroid hormones. The present investigation indicates that dietary vitamin E may induce changes in stereoidogenesis by affecting cholesterol homeostasis.

Androgen metabolism in the Australian lizard Tiliqua rugosa

Nonmammalian vertebrates possess some unusual features in their hormonal systems/ when compared to mammals. As a consequence, they can make an important contribution in investigations concerning the fundamental mechanisms operating in endocrinology. Such studies concerning androgens include inter alia their effects on developmental aspects in the brain of birds and related singing behaviour; the role of neural enzymes in reproductive processes in fish; and the relation between androgens and the stages of spermatogenesis in amphibia, The present thesis examines the biochemistry of androgens in the Australian lizard Tiliqua rugosa. The major compounds studied were testosterone and epitestosterone, which are known to be present in high concentrations in the plasma of the male animal. Previous investigations are expanded, particularly in the areas of steroid identification and testicular biosynthesis. In addition, preliminary studies on the metabolism in the brain (and other tissues) and plasma protein binding are reported. The presence of epitestosterone as a major free androgen in the plasma of the male lizard was confirmed. Other steroids were found in the sulphate fraction. Testosterone sulphate was the most rigorously identified compound, while some evidence was also found for the presence of conjugated 5-androstene-3β,17-diols, etiocholanolone and dehydroepiandrosterone (DHA). Epitestosterone does not appear to be extensively conjugated in this animal. Steroids were not found to be conjugated as glucuronides. The identification studies employed a novel method of electrochemical detection of steroids. This technique was investigated and extended in the current thesis. Biosynthetic studies were carried out on androgen interconversions in the testis, in vitro. The major enzyme activities detected were 17α-arid 17β-oxidoreductases (17α-OR and l7β-OR) and 3β-hydroxysteroid dehydrogenase (3β-HSD)/isonerase. No evidence was found for the presence of a steroid-17-epimerase that would directly interconvert testosterone and epitestosterone. The 17-oxidoreductases were found to be dependent on the cofactor NBDFH. Testosterone appears to be formed mainly via the 4-ene pathway, whereas epitestosterone is formed from both the 4- and 5-ene routes. The compound 5-androstene-3β, 17α-diol was found to be an intermediate in the synthesis of epitestosterone from DHA. Temperature was found to significantly affect 17α-OR activity (maximum at 32°C). In contrast,17β-OR activity was independent of this factor in the testis. Androgen metabolism in the testis was found to be regulated by cofactors, temperature and season. The major enzyme activities found in the male brain were 17α- and 17β-OR. 3βHSD/isomerase was not found; however a low activity of 5α-reductase was identified. Aromatase activity was not positively identified, but preliminary results suggest that it may be present at low levels. The 17-oxidoreductases were widespread throughout the brain. The 17α-OR was significantly lower in the forebrain than other brain sections. The 170-OR activity did not vary significantly throughout the organ, although there was a trend for its activity to be higher in the midbrain region (containing the hypothalamus in these sections). The concentration of endogenous steroids in brain tissue was estimated by radioimmunoassay. Epitestosterone was found throughout the organ structure, whereas testosterone was found mainly in the midbrain (containing hypothalamic regions in these sections). Correlations between enzyme activities and steroid concentrations in brain regions suggested that the main function of 17α-OR is to produce epitestosterone, whereas the 17β-OR may catalyse a more reversible reaction in vivo. Temperature was found to significantly affect both 17α- and 17β-OR activities in the brain. In contrast to the testis, the maximum activity of the brain enzymes occurred at 37°C. The level of 17α-OR activity in the male lizard (100 nmol/g tissue/h) is the highest reported for this enzyme in vertebrates. Both activities were found to be quantitatively similar in the whole brain homogenates of male and female animals, and did not vary seasonally when examined in the male. The 17-oxidoreductases were also found in most other tissues in T.rugosa, including epididymis, adrenal, kidney and liver (but not blood). This suggests that the high activities of both 17α-OR and 17β-OR are dominant features of the steroid system in this animal. The formation of 11-oxygenated compounds was found in the adrenal, in addition to the formation of polar metabolites in the kidney and liver (possibly polyhydroxylated and conjugated steroids). A preliminary investigation into the plasma binding of androgens was carried out. The insults suggest that there are several binding sites for testosterone; one with high affinity and low capacity; the other with low affinity and high capacity. Binding experiments were carried out at 32°C. At this temperature, specific binding was greater than at 25 or 37°C. From the results of competition studies it was suggested that epitestosterone (with a K(i)= 3 X 10 (-6)M for testosterone binding) regulates the binding of testosterone (K(i)=10(-7)M) and hence the concentrations of the latter steroid as a free compound in plasma. In general, the study has shown that the biochemistry of androgens in the reptile T.rugosa is largely similar to that found in other vertebrates. The major difference is a greatly increased activity of 17α-OR, which causes a higher concentration of 17α-compounds to be present in the tissues of this lizard. The physiological roles for epitestosterone are not yet clear. However it appears from this study that this steroid regulates testosterone concentrations in several tissues by either steroidogenic or binding mechanisms. Several major influences on this regulation include temperature, availability of cofactors and seasonal effects.

Stimulation of apolipoprotein D secretion by steroids coincides with inhibition of cell proliferation in human LNCaP prostate cancer cells

Although steroid hormones are known to play a predominant role in the regulation of cell growth in hormone-sensitive cancers, their mechanisms of action, especially their interaction with growth factors and/or growth inhibitors, is poorly understood. We have recently observed that the effects of androgens and estrogens on the expression of the major protein found in human breast gross cystic disease fluid, protein-24, are opposite to their respective action on cell proliferation in human breast cancer cell lines. Somewhat surprisingly, the recent elucidation of the amino acid sequence of this progesterone binding protein reveals that this tumor marker is apolipoprotein D (apo D), a member of a superfamily of lipophilic ligand carrier proteins. The present study was designed to determine whether apo D is secreted by human prostate cancer cells and could thus be a new marker of steroid action in these cancer cells, and whether the sex steroid-induced stimulation of apo D secretion coincides with inhibition of cell proliferation. We took advantage of the biphasic pattern of the effect of steroids on the proliferation of the human prostate cancer LNCaP cell line, which offers the opportunity to discriminate between positive and negative steroid receptor-regulated cell growth processes. A 10-day exposure to low concentrations of dihydrotestosterone and testosterone caused a potent stimulation of LNCaP cell proliferation, whereas incubation with higher concentrations of these androgens led to a progressive decrease in cell proliferation towards basal levels. The biphasic action of androgens was also observed on apo D secretion, the effects on apo D secretion being inversely related to their action on LNCaP cell proliferation. Similar opposite biphasic effects were also observed with 9 other steroids, thus indicating that the stimulation of secretion of this new biochemical marker coincides with inhibition of cell proliferation in LNCaP human prostatic cancer cells.

Androgen-regulated metabolism and biosynthesis in prostate cancer

Metabolic changes are a well-described hallmark of cancer and are responses to changes in the activity of diverse oncogenes and tumour suppressors. For example, steroid hormone biosynthesis is intimately associated with changes in lipid metabolism and represents a therapeutic intervention point in the treatment of prostate cancer (PCa). Both prostate gland development and tumorigenesis rely on the activity of a steroid hormone receptor family member, the androgen receptor (AR). Recent studies have sought to define the biological effect of the AR on PCa by defining the whole-genome binding sites and gene networks that are regulated by the AR. These studies have provided the first systematic evidence that the AR influences metabolism and biosynthesis at key regulatory steps within pathways that have also been defined as points of influence for other oncogenes, including c-Myc, p53 and hypoxia-inducible factor 1α, in other cancers. The success of interfering with these pathways in a therapeutic setting will, however, hinge on our ability to manage the concomitant stress and survival responses induced by such treatments and to define appropriate therapeutic windows.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

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

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

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

The methyl-CpG-binding protein MECP2 is required for prostate cancer cell growth.

The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.

Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats

Advanced glycation end products (AGEs), formed from the nonenzymatic glycation of proteins and lipids with reducing sugars, have been implicated in many diabetic complications; however, their role in diabetic retinopathy remains largely unknown. Recent studies suggest that the cellular actions of AGEs may be mediated by AGE-specific receptors (AGE-R). We have examined the immunolocalization of AGEs and AGE-R components R1 and R2 in the retinal vasculature at 2, 4, and 8 months after STZ-induced diabetes as well as in nondiabetic rats infused with AGE bovine serum albumin for 2 weeks. Using polyclonal or monoclonal anti-AGE antibodies and polyclonal antibodies to recombinant AGE-R1 and AGE-R2, immunoreactivity (IR) was examined in the complete retinal vascular tree after isolation by trypsin digestion. After 2, 4, and 8 months of diabetes, there was a gradual increase in AGE IR in basement membrane. At 8 months, pericytes, smooth muscle cells, and endothelial cells of the retinal vessels showed dense intracellular AGE IR. AGE epitopes stained most intensely within pericytes and smooth muscle cells but less in basement membrane of AGE-infused rats compared with the diabetic group. Retinas from normal or bovine-serum-albumin-infused rats were largely negative for AGE IR. AGE-R1 and -R2 co-localized strongly with AGEs of vascular endothelial cells, pericytes, and smooth muscle cells of either normal, diabetic, or AGE-infused rat retinas, and this distribution did not vary with each condition. The data indicate that AGEs accumulate as a function of diabetes duration first within the basement membrane and then intracellularly, co-localizing with cellular AGE-Rs. Significant AGE deposits appear within the pericytes after long-term diabetes or acute challenge with AGE infusion conditions associated with pericyte damage. Co-localization of AGEs and AGE-Rs in retinal cells points to possible interactions of pathogenic significance.

The ETS family member GABPα modulates androgen receptor signalling and mediates an aggressive phenotype in prostate cancer

In prostate cancer (PC), the androgen receptor (AR) is a key transcription factor at all disease stages, including the advanced stage of castrate-resistant prostate cancer (CRPC). In the present study, we show that GABPα, an ETS factor that is up-regulated in PC, is an AR-interacting transcription factor. Expression of GABPα enables PC cell lines to acquire some of the molecular and cellular characteristics of CRPC tissues as well as more aggressive growth phenotypes. GABPα has a transcriptional role that dissects the overlapping cistromes of the two most common ETS gene fusions in PC: overlapping significantly with ETV1 but not with ERG target genes. GABPα bound predominantly to gene promoters, regulated the expression of one-third of AR target genes and modulated sensitivity to AR antagonists in hormone responsive and castrate resistant PC models. This study supports a critical role for GABPα in CRPC and reveals potential targets for therapeutic intervention.

Maintaining and reprogramming genomic androgen receptor activity in prostate cancer

Prostate cancer treatment is dominated by strategies to control androgen receptor (AR) activity. AR has an impact on prostate cancer development through the regulation of not only transcription networks but also genomic stability and DNA repair, as manifest in the emergence of gene fusions. Whole-genome maps of AR binding sites and transcript profiling have shown changes in the recruitment and regulatory effect of AR on transcription as prostate cancer progresses. Defining other factors that are involved in this reprogramming of AR function gives various opportunities for cancer detection and therapeutic intervention.

N-linked glycosylation supports cross-talk between receptor tyrosine kinases and androgen receptor

Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs). In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP) than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R) ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an important challenge in the future.

The androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man

The androgen receptor (AR) regulates prostate cell growth in man, and prostate cancer is the commonest cancer in men in the UK. We present a comprehensive analysis of AR binding sites in human prostate cancer tissues, including castrate-resistant prostate cancer (CRPC). We identified thousands of AR binding sites in CRPC tissue, most of which were not identified in PC cell lines. Many adjacent genes showed AR regulation in xenografts but not in cultured LNCaPs, demonstrating an in-vivo-restricted set of AR-regulated genes. Functional studies support a model of altered signaling in vivo that directs AR binding. We identified a 16 gene signature that outperformed a larger in-vitro-derived signature in clinical data sets, showing the importance of persistent AR signaling in CRPC.

Chromatin binding by the androgen receptor in prostate cancer

Alterations in transcriptional programs are fundamental to the development of cancers. The androgen receptor is central to the normal development of the prostate gland and to the development of prostate cancer. To a large extent this is believed to be due to the control of gene expression through the interaction of the androgen receptor with chromatin and subsequently with coregulators and the transcriptional machinery. Unbiased genome-wide studies have recently uncovered the recruitment sites that are gene-distal and intragenic rather than associated with proximal promoter regions. Whilst expression profiles from AR-positive primary prostate tumours and cell lines can directly relate to the AR cistrome in prostate cancer cells, this distribution raises significant challenges in making direct mechanistic connections. Furthermore, extrapolating from datasets assembled in one model to other model systems or clinical samples poses challenges if we are to use the AR-directed transcriptome to guide the development of novel biomarkers or treatment decisions. This review will provide an overview of the androgen receptor before addressing the challenges and opportunities created by whole-genome studies of the interplay between the androgen receptor and chromatin.

Global identification of androgen response elements

Chromatin immunoprecipitation (ChIP) is an invaluable tool in the study of transcriptional regulation. ChIP methods require both a priori knowledge of the transcriptional regulators which are important for a given biological system and high-quality specific antibodies for these targets. The androgen receptor (AR) is known to play essential roles in male sexual development, in prostate cancer and in the function of many other AR-expressing cell types (e.g. neurons and myocytes). As a ligand-activated transcription factor the AR also represents an endogenous, inducible system to study transcriptional biology. Therefore, ChIP studies of the AR can make use of treatment contrast experiments to define its transcriptional targets. To date several studies have mapped AR binding sites using ChIP in combination with genome tiling microarrays (ChIP-chip) or direct sequencing (ChIP-seq), mainly in prostate cancer cell lines and with varying degrees of genomic coverage. These studies have provided new insights into the DNA sequences to which the AR can bind, identified AR cooperating transcription factors, mapped thousands of potential AR regulated genes and provided insights into the biological processes regulated by the AR. However, further ChIP studies will be required to fully characterise the dynamics of the AR-regulated transcriptional programme, to map the occupancy of different AR transcriptional complexes which result in different transcriptional output and to delineate the transcriptional networks downstream of the AR.