Steroid Precursors Research Articles

Complete genome sequence of ‘Mycobacterium neoaurum’ NRRL B-3805, an androstenedione (AD) producer for industrial biotransformation of sterols

Microbial bioconversion of sterols into high value steroid precursors, such as 4-androstene-3,17-dione (AD), is an industrial challenge. Genes and enzymes involved in sterol degradation have been proposed, although the complete pathway is not yet known. The genome sequencing of the AD producer strain ‘Mycobacterium neoaurum’ NRRL B-3805 (formerly Mycobacterium sp. NRRL B-3805) will serve to elucidate the critical steps for industrial processes and will provide the basis for further genetic engineering. The genome comprises a circular chromosome (5 421 338bp), is devoid of plasmids and contains 4844 protein-coding genes.

Determination of a steroid profile in heel prick blood using LC-MS/MS.

The aim of this study was to improve the sensitivity of the congenital adrenal hyperplasia (CAH) neonatal screening by including second-tier steroid profiling on a DBS using LC-MS. We developed a method to measure the steroid profile in DBS and established gestational age-specific reference ranges of cortisol, cortisone, 11-deoxycortisol, 21-deoxycortisol, 17-hydroxyprogesterone, testosterone, Δ4-androstenedione, corticosterone and 11-deoxycorticosterone using 450 heel prick samples of neonates, participating in the Dutch Screening Program. Analyzing 92 cards with a positive CAH screening showed that only 21-deoxycortisol was 100% specific for diagnosed CAH patients. Steroid precursors can be measured in DBS and we suggest to implement the method as a second tier testing for CAH in The Netherlands.

Sexually Diergic Trophic Effects of Estradiol Exposure on Developing Bovine Cerebellar Granule Cells

In the mammalian brain, the differentiation of neural cells and the developmental organization of the underlying circuitry are influenced by steroid hormones. The estrogen 17-β estradiol (E2) is one of the most potent regulators of neural growth during prenatal life, synthetized locally from steroid precursors including prenatal testicular testosterone. Estradiol promotes brain differentiation counting sexually dimorphic neural circuits by binding to the estrogen receptors, ER-α and ER-β. The cerebellum has been described as a site of estrogen action and a potentially sexually dimorphic area. The goal of this study was to analyze the capacity of E2 to affect the growth of male and female fetal bovine cerebellar granule. We performed primary cultures of fetal cerebellar granules, and verified the mRNA expression of the ER-α and ER-β in both sexes. Moreover, the distribution of ERs in the male and female cerebellar granules of the second fetal stage was characterized by immunohistochemistry. We measured morphological parameters in presence (or absence) of estradiol administration, focusing on the variations of the dendritic branching pattern of granule neurons. By using the nonparametric combination and permutation testing approach, we proposed a sophisticated multivariate statistical analysis to demonstrate that E2 induces multifarious and dimorphic changes in the granule cells. E2 exerts trophic effects in both female and male granules and this effect is stronger in female. Male granules treated with E2 became similar to female control granule. Bos taurus species has a long gestation and a large brain that offers an interesting alternative in comparative neuroscience.

Difference in expression patterns of placental cholesterol transporters, ABCA1 and SR-BI, in Meishan and Yorkshire pigs with different placental efficiency.

Cholesterol is a key cell membrane component and precursor of steroid hormones. The maternal cholesterol is an important exogenous cholesterol source for the developing embryos and its transportation is mediated by ABCA1 and SR-BI. Here we reported that during the peri-implantation period in pigs, ABCA1 was expressed by uterine luminal epithelium (LE) and interestingly, its expression was more abundantly in LE on mesometrial side of uterus. However, SR-BI was expressed primarily by LE, glandular epithelial cells (GE) and trophoblast cells (Tr). During the placentation period, the expression levels of ABCA1 and SR-BI proteins at epithelial bilayer and placental areolae were significantly higher in Chinese Meishan pigs compared to Yorkshire pigs. Consisitently, mRNA levels of HMGCR, the rate-limiting enzyme for cholesterol synthesis, were significantly higher in Meishan placentas than in Yorkshire placentas. Our findings revealed the routes of transplacental cholesterol transport mediated by ABCA1 and SR-BI in pigs and indicated that ABCA1 related pathway may participate in anchoring the conceptus to the mesometrial side of uterus. Additionally, an ABCA1 dependent compensatory mechanism related to the placental efficiency in response to the smaller placenta size in Meishan pigs was suggested.

Association Between Serum Levels of Testosterone and Estradiol With Meibomian Gland Assessments in Postmenopausal Women

PurposeThe aims of this analysis were to determine if there is an association between serum levels of testosterone and estradiol with meibomian gland (MG) morphology and lipid layer thickness.MethodsThe data used for this analysis were collected from postmenopausal women with and without dry eye disease. Meibography was used to assess MG dropout on the central two-thirds of the eyelid and biomicroscopy was used for assessing MG expressibility and meibum quality. Venous blood samples were drawn for serum hormone level analysis. The Kruskal-Wallis test and Spearman correlations were used for statistical analysis.ResultsOne hundred ninety-eight postmenopausal women with an average age of 61.2 (±9.1) years were included in this analysis. Testosterone levels showed significant differences between MG dropout grades 1 and 4 (P = 0.002) and grades 2 and 4 (P = 0.01), whereas estradiol levels were different based on MG dropout (P = 0.53). No significant correlations were found between testosterone (r = 0.10, P = 0.17) or estradiol (r = 0.05, P = 0.45) and lipid layer thickness.ConclusionsTestosterone levels were increased with MG dropout, which was significant between the mild and severe dropout groups, whereas no significant differences were found with estradiol and any MG assessment. Although the literature suggests an association of serum hormone levels and pathogenesis of dry eye disease in postmenopausal women, analysis of active sex steroid precursors and local tissue hormone levels may prove more useful.

MCL1 is a key regulator of steroidogenesis in mouse Leydig cells.

Myeloid cell leukemia-1 (MCL1), an anti-apoptotic member of the BCL2 family, is expressed abundantly in the testis. Previous characterization revealed that MCL1 is expressed exclusively in the Leydig cells in the mouse testis, yet what it does in these cells remains unknown. We therefore analyzed testosterone biosynthesis in isolated primary Leydig cells and the MA-10 cell line, in which MCL1 was knocked down using an siRNA strategy. The mRNA abundance of the steroidogenic genes Star, Cyp11a1, Cyp17a1, Hsd3b1, Srd5a, and the luteinizing hormone/choriogonadotropin receptor Lhcgr were significantly reduced following MCL1 knockdown. Of the two enzymes required for testosterone biosynthesis, STAR and P450 SCC (encoded by Cyp11a1) enzyme abundance was also reduced following Mcl1 siRNA treatment, possibly leading to the reduced production of sex steroid precursors, and testosterone in these knockdown cells. Despite its classification as an anti-apoptosis protein, Mcl1 siRNA treatment did not affect cell survival. Collectively, our findings indicate that MCL1 plays a pivotal role in Leydig-cell steroidogenesis, and might provide novel insights into metabolic regulation in this cell. Mol. Reprod. Dev. 83: 226-235, 2016. © 2016 Wiley Periodicals, Inc.

Associations between peripheral androgens and cortisol in infertile women

Testosterone has in recent years been proven essential for normal growth and maturation of small growing follicles. Concomitantly, low functional ovarian reserve (LFOR), characterized by a small growing follicle pool, has been associated with low testosterone levels, which can be of ovarian and/or adrenal origin. In this study we, therefore, investigated whether peripheral sex steroid precursors and testosterone levels potentially reflect on adrenal function. In a retrospective cohort study of 355 consecutive infertile women, who presented to an academically affiliated fertility center in New York City, we investigated in a series of statistical models whether low peripheral sex steroid precursors and testosterone are associated with peripheral cortisol (C) levels, reflecting adrenal function. To determine potential correlations, we investigated the dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), androstenedione (AD), total testosterone (TT), free testosterone (FT); sex hormone binding globulin (SHBG), anti-Müllerian hormone (AMH), thyroid stimulating hormone (TSH) and C in a series of multivariate and logistic regression analyses, utilizing C either as a continuous variable or with cut off <5.0μg/dL, and TT only as a continuous variable. Practically all models demonstrated significant predictability of peripheral sex hormone precursors for C levels, with DHEA demonstrating the strongest and most consistent predictability as an individual parameter and as part of the DHEAS/DHEA ratio. We conclude that in infertile women peripheral sex hormone precursors, especially DHEA, reflect C levels and, therefore, adrenal function. In infertile women, at all ages low levels of sex hormone precursors, therefore, should be considered indications for further adrenal assessments.

A dual stable isotope method by LC-MS/MS to define patterns of androgen metabolism in localized versus advanced prostate cancer.

40 Background: Prior work has shown that androstenedione (AD) rather than testosterone (T) is the preferred substrate of 5α-reductase for dihydrotestosterone (DHT) synthesis in castration-resistant prostate cancer. However, patterns of metabolism in hormone-naive prostate cancer are still poorly defined. Previously, we reported on the utility of dual radioisotope labeling of steroid precursors to characterize androgen metabolism in localized prostate tissue. We now describe an alternative approach via stable isotope labeling and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which has unique advantages over the former method. Methods: LNCaP cell lines and prostate tissue from patients undergoing radical prostatectomy for localized cancer were incubated in serum-free media, spiked with 13C-labeled AD and 2H-labeled T. Media was collected at 7, 24, and 48 hours of incubation. Steroids were extracted, separated, and then analyzed by way of LC-MS/MS to identify labeled metabolites of AD and T. Results: Incubation of labeled AD and T resulted in conversion over time to both 13C-labeled and 2H-labeled downstream metabolites, 5α-dione and DHT. Although both precursors contributed to 5α-dione and DHT formation, the steroid of origin could be determined on the basis of differential labeling. In ex-vivo tissue incubations, unlabeled 5α-dione was also observed, which was distinguishable from its stable isotopic form and most likely represents endogenous steroid. Conclusions: Both cell lines and tissue appear to metabolize labeled AD and T, and formation of DHT occurs through both precursors. Furthermore, the presence of endogenous 5α-dione suggests that alternative pathways of DHT synthesis, which bypass T, may be naturally accessible in the hormone-naïve setting. We propose a robust ex-vivo technique to readily track simultaneous pathways of DHT synthesis in prostate cancer. Future work will focus on defining metabolic phenotypes of localized prostate cancer and specific patterns of flux under different hormonal and pharmacologic conditions.

Cholesterol and atherosclerosis. Historical considerations and treatment

Cholesterol is a precursor of steroid hormones and an essential component of the cell membrane, however, altered regulation of the synthesis, absorption and excretion of cholesterol predispose to cardiovascular diseases of atherosclerotic origin. Despite, the recognition of historical events for 200 years, starting with Michel Chevreul naming «cholesterol»; later on, Lobstein coining the term atherosclerosis and Marchand introducing it, Anichkov identifying cholesterol in atheromatous plaque, and Brown and Goldstein discovering LDL receptor; as well as the emerging of different drugs, such as fibrates, statins and cetrapibs this decade, promising to increase HDL and the most recent ezetimibe and anti-PCSK9 to inhibit the degradation of LDL receptor, however morbidity has not been reduced in cardiovascular disease.

Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1.

The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7β-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0.

Dehydroepiandrosterone Alters Retinol Status and Expression of the β-Carotene 15,15'-Monooxygenase and Lecithin:Retinol Acyltransferase Genes.

Dehydroepiandrosterone (DHEA) and its sulfate ester DHEA-sulfate (DHEA-S) are the most abundant adrenal steroids in humans. DHEA has a critical role as a steroidal precursor of androgens and/or estrogens, and in human studies and animal experiments, both DHEA and DHEA-S have multiple beneficial effects. However, there are few reports regarding the relationship between DHEA and nutrient status, especially for vitamins. Therefore, we elucidated the effect of DHEA administration on retinol status. Wistar rats were fed with a standard diet containing 0.4% (wt/wt) DHEA for 2 wk. We assessed retinol status and the expression of retinol-related proteins, including metabolic enzymes, binding proteins, cytochrome P450 (CYP) enzymes, and antioxidant enzymes. Retinol levels in the plasma and the liver of DHEA-fed rats were lower than those of controls. Expression of β-carotene 15,15'-monooxygenase (BCMO) in the liver and intestine of DHEA-fed rats was lower, whereas BCMO expression in the testes of DHEA-fed rats was higher than that of controls. Expression of the retinol-metabolizing aldehyde dehydrogenase (ALDH) enzyme ALDH1A2 was repressed in the liver of DHEA rats, whereas ALDH1A1 expression was unaltered. Hepatic expression of lecithin:retinol acyltransferase (LRAT) and CYP26A1 was lower in DHEA-fed rats than in controls. Retinol status in DHEA-fed rats might be affected by altered BCMO expression in the liver and intestine and hepatic LRAT expression, whereas BCMO expression in peripheral tissues may be regulated in a tissue-specific manner. We have shown that DHEA administration may influence retinol status and the expression of retinol-related proteins.

Innovative Target Therapies Are Able to Block the Inflammation Associated with Dysfunction of the Cholesterol Biosynthesis Pathway.

The cholesterol pathway is an essential biochemical process aimed at the synthesis of bioactive molecules involved in multiple crucial cellular functions. The end products of this pathway are sterols, such as cholesterol, which are essential components of cell membranes, precursors of steroid hormones, bile acids and other molecules such as ubiquinone. Several diseases are caused by defects in this metabolic pathway: the most severe forms of which cause neurological involvement (psychomotor retardation and cerebellar ataxia) as a result of a variety of cellular impairments, including mitochondrial dysfunction. These pathologies are induced by convergent mechanisms in which the mitochondrial unit plays a pivotal role contributing to defective apoptosis, autophagy and mitophagy processes. Unraveling these mechanisms would contribute to the development of effective drug treatments for these disorders. In addition, the development of biochemical models could have a substantial impact on the understanding of the mechanism of action of drugs that act on this pathway in multifactor disorders. In this review we will focus in particular on inhibitors of cholesterol synthesis, mitochondria-targeted drugs and inhibitors of the inflammasome.

74 ANALYSIS OF STEROID HORMONES IN BOVINE OVIDUCTAL FLUID BY GAS CHROMATOGRAPHY COUPLED WITH TANDEM MASS SPECTROMETRY

Steroid hormones play key roles in the regulation of physiological changes in the mammalian genital tract, including the oviduct. The aim of this study was to determine the variations in steroid hormone concentrations in bovine oviducal fluid (OF) during the oestrous cycle. Bovine oviducts were collected at a local slaughterhouse and classified into 4 stages according to the ovarian and corpus luteum (CL) morphologies (n = 18–25 cows/stage): post-ovulatory (postov), mid luteal (mid-lut), late luteal (late-lut), and preovulatory (preov). Follicular fluid was also collected from the preov follicles. Animals with follicular oestradiol-17β (E2) &lt;40 ng mL–1 or progesterone (P4) &gt;160 ng mL–1 (cystic follicles) or E2 : P4 ratio &lt;1 (atretic follicles) were excluded. Oviducal fluids were collected from contra- and ipsilateral (to the CL) ampullas by squeezing. They were then centrifuged and stored at –80°C. Steroids were extracted from pools of 150 to 200 µL of OF (4–10 cows/pool; 3–4 pools per “stage × side” group), purified, fractioned by high-performance liquid chromatography, derivatized, and analysed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Comparisons between groups were made with a two-way ANOVA followed by Bonferroni post-tests. Data are expressed as means ± SEM. A P-value &lt; 0.05 was considered as significant. In ipsilateral OF, the concentrations of P4 increased from postov (56.9 ± 13.4 ng mL–1) to mid-lut (120.3 ± 34.3 ng mL–1; P &lt; 0.01) then decreased from late-lut (76.7 ± 1.8 ng mL–1) to preov (6.3 ± 1.7 ng mL–1; P &lt; 0.001) stages, and were 4 to 16 times more concentrated than in contralateral OF. Several P4 reduced metabolites (5α-dihydroP4, 20α-dihydroP4, 17α-hydroxyP4, 3α5α20α-hexahydroP4, 3β5α20α-hexahydroP4) followed the same pattern of variation. Concentrations of the P4 precursor pregnenolone were highest at postov (4.2 ± 0.3 ng mL–1 in ipsilateral OF) then decreased from mid-lut to preov (3.4 ± 0.6 to 1.3 ± 0.1 ng mL–1; P &lt; 0.001) and were higher than in contralateral OF at postov (P &lt; 0.001) and mid-lut (P &lt; 0.01) stages. Concentrations of E2 were higher in preov (290.5 ± 63.2 pg mL–1 in ipsilateral OF) compared to all other stages (P &lt; 0.05) with no difference between ipsi- and contralateral sides at any stage. Concentrations of the oestrogen precursors androstenedione and testosterone displayed the same pattern of variation as E2. In contrast, concentrations of dehydroepiandrosterone and estrone did not vary between stages or sides relative to the CL. In addition, high concentrations of cortisol and cortisone were found in ipsi- and contralateral OF at all stages of the oestrous cycle (on average 48.6 and 31.9 ng mL–1, respectively). In conclusion, numerous steroids, steroid precursors, and metabolites were found in the bovine OF. The local transfer of ovarian steroids creates a highly concentrated and fluctuating hormonal environment in the ipsilateral OF during the oestrous cycle. These results could be useful to improve media for IVF, development, and oviducal cell culture.

Neuroprotective properties of dehydroepiandrosterone-sulfate and its relationship to interleukin 6 after aneurysmal subarachnoid hemorrhage: a prospective cohort study.

IntroductionThe established neuroprotective property of the sex steroid precursor dehydroepiandrosterone-sulfate (DHEAS) has not yet been investigated in the context of aneurysmal subarachnoid hemorrhage (aSAH). The influence of DHEAS on inflammatory response resulting in modulation of interleukin 6 (IL-6) synthesis has been shown. Here, we evaluate DHEAS serum levels after aSAH (day 0–14) and levels of IL-6 related to functional outcome at discharge and at six months.MethodsA complete data set (DHEAS and IL-6 serum levels for days 0, 1, 4, 7, 10 and 14 after aSAH) and outcome assessment at discharge according to modified Rankin Scale score (mRS) was available for 53 patients of the initially screened cohort (n = 109). Outcome assessment six months after aSAH was obtained from 41 patients. Logarithmized levels of DHEAS and IL-6 were related to dichotomized functional outcome either assessed at discharge or at six months. A mixed between-within subjects ANOVA was applied for statistical analysis (SPSS 21.0).ResultsDHEAS and IL-6 levels across time were related to functional outcome. Regarding outcome assessment at discharge and at six months after aSAH, DHEAS levels (transformed to square root for statistical purposes) were considerably higher in patients with favorable outcome (mRS 0–2) (p = .001; p = .020). Inversely, in patients with favorable outcome either at discharge or six months after aSAH, lower IL-6 levels (logarithmized for statistical purposes) were observed across time (both p < .001).ConclusionWe provide new evidence that DHEAS is associated with protective properties resulting in improvement of functional outcome after aSAH, possibly by influencing the inflammatory response after aSAH shown in the decreasing IL-6 serum levels. But the results for outcome six months after SAH are limited due to a high drop-out rate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0954-1) contains supplementary material, which is available to authorized users.

Abstract C97: Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling

Abstract Introduction Castration resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, driven by adrenal precursors and potentially de novo steroid synthesis in other organ tissues including prostate. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1 and the AR has been demonstrated to prolong survival of CRPC patients. In this study, we created a co-culture model using human prostate and adrenal tumours to study abiraterone resistance. Materials and Methods Human androgen-dependent PC (VCaP) and CPRC clones were cultured with substrates for de novo androgen synthesis or with adrenal androgens, or cultured with human adrenal cells (H295R) and treated with either the CYP17A1 inhibitor abiraterone or the antiandrogen MDV3100. Male mice bearing VCaP tumours and human adrenal H295R xenografts were castrated and treated with placebo or abiraterone. Tumour response was assessed by tumour growth, PSA release, steroid quantitation by (LC/MS-MS), immunohistochemistry and mRNA expression analysis of steroidogenic enzymes and nuclear receptors. Results In vitro, physiological levels of adrenal androgen precursors DHEA and androstenedione induced cell growth in parental and CRPC VCaP sub clones, whereas precursor steroids pregnenolone and progesterone for de novo synthesis did not. In a co-culture model, abiraterone blocked H295R-induced growth of VCaP cells. Likewise, in vivo, H295R tumours stimulated castration-resistant VCaP growth. This stimulative effect was inhibited by abiraterone, reducing - but not fully blocking - growth and PSA production. In the absence of H295R tissue, VCaP xenografts grew slow but became castration resistant nonetheless. In contrast to the observed effects on VCaP growing in castrate animals bearing H295R tumours, abiraterone was unable to inhibit the slow VCaP growth and low PSA production in castrate mice without H295R xenografts. LC/MS-MS analysis of plasma and tumour tissue could not confirm increased de novo production of androgens. Castrate and abiraterone-resistant VCaP tumours were characterised by increased levels of AR, AR variants and glucocorticoid receptor (GR) expression, resulting in equal AR target gene expression levels. Conclusions Our data indicate that AR ligand dependent regrowth of CRPC is predominantly supported via adrenal steroid production. Abiraterone resistant disease of VCaP relies on AR overexpression, expression of ligand independent AR variants and GR signalling. Citation Format: Jan Matthijs Moll, Johannes Hofland, Wilma Teubel, Corrina M.A. de Ridder, Anne E. Taylor, Ralph Graeser, Wiebke Arlt, Guido W. Jenster, Wytske M. van Weerden. Beyond intratumoural steroidogenesis: abiraterone resistance mediated by AR variants and glucocorticoid receptor signalling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C97.

Regulation of human (adrenal) androgen biosynthesis—New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

Clinical significance of D4A in prostate cancer therapy with abiraterone

Androgen deprivation therapy (ADT), either by surgical or medical castration depletes testicular production of testosterone (T) and has long been considered as the mainstay of upfront treatment for advanced prostate cancer. Despite the initial response to this treatment, disease progression to castration-resistant prostate cancer (CRPC) usually occurs. The adrenal production of androgen precursor steroids, such as dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), permits intratumoral production of T and 5α-dihydrotestosterone (DHT), activation of androgen receptor (AR) and AR-dependent gene expression. Abiraterone (Abi; given as Abi acetate) and enzalutamide (Enz) are 2 of the agents that target different parts of the androgen pathway by blocking androgen production and signaling, ultimately improving overall survival for patients with metastatic CRPC.1,2 Abi is a potent inhibitor of 17α-hydroxylase/17,20-lyase (CYP17A1), which is a key enzyme required for DHEA and DHEA-S production. Synthesis of DHT, the most potent androgen, from DHEA necessitates a series of enzymes, including 3β-hydroxysteroid dehydrogenase (3βHSD), steroid-5α-reductase (SRD5A), and 17β-hydroxysteroid dehydrogenase (17βHSD) isoenzymes. This process enables the conversion of ADT-responsive prostate cancer into CRPC. Multiple retrospective reviews have shown the limited efficacy of Enz after Abi treatment failure and vice versa.3,4 To date there are very little data on the appropriate sequence of Enz and Abi utilization, which might thereby maximize the potential efficacy and duration of response to treatment with these agents. Our recently published research demonstrates that Δ4, 3-keto-abi (D4A) is a metabolite of Abi that is formed in patients with prostate cancer and has CYP17A1 inhibition activity that is comparable to Abi.5 Furthermore, D4A inhibits 3βHSD (a key enzyme involved in CRPC progression) more potently than Abi and at higher concentrations it demonstrates significant SRD5A inhibitory effects as well. Moreover, although Abi has been shown to have AR antagonistic activity,6 we observed more potent AR inhibition activity with D4A that is comparable to Enz in our experiments.5 As a result, by targeting multiple steps of the AR signaling pathway, D4A showed a significant increase in progression free survival compared to Abi and Enz in our xenograft models, suggesting that it might play an important role in the clinical activity of Abi in CRPC patients. Abi is converted to D4A by 3βHSD. Increased 3βHSD enzymatic function, with the HSD3B1(1245C) genetic variant encoding a missense in 3βHSD1 as one known mechanism, also increases intratumoral flux to DHT that may result in the development of treatment resistance.7 Therefore, D4A conversion ratio could serve as an indicator of 3βHSD activity and ultimately may predict resistance to hormonal therapy for prostate cancer. On the other hand, higher 3βHSD activity would translate into a higher D4A concentration, which is a more potent metabolite than Abi as described above. Although the Abi to D4A conversion ratio is low (at about 5%) in most patients, it appears to be quite variable. A higher conversion ratio might mimic conditions of treatment with Enz, particularly given the AR antagonist activity of D4A. In other words, 3βHSD activity level in a patient might be helpful in predicting the potential response to Abi and ultimately a patient with higher concentration of D4A with Abi treatment might be less likely to benefit from subsequent Enz treatment. Conversely, increasing D4A levels by increasing Abi concentrations or the conversion to D4A might increase the clinical benefit of treatment with Abi. In addition, because 3βHSD is required for mineralocorticoid synthesis, 3βHSD inhibition by D4A might reduce mineralocorticoid production that could translate into a better side effect profile (less hypertension and hypokalemia) and potentially lower dose of prednisone requirement in patients with higher D4A levels. These questions have yet to be answered in clinical studies. Finally, it is possible that steroidogenic metabolites of Abi are not limited to D4A, and that other metabolites with clinically relevant biochemical activity contribute to response and resistance to treatment with Abi.

Computational analysis of liquid chromatography-tandem mass spectrometric steroid profiling in NCI H295R cells following angiotensin II, forskolin and abiraterone treatment

Adrenal steroid hormones, which regulate a plethora of physiological functions, are produced via tightly controlled pathways. Investigations of these pathways, based on experimental data, can be facilitated by computational modeling for calculations of metabolic rate alterations. We therefore used a model system, based on mass balance and mass reaction equations, to kinetically evaluate adrenal steroidogenesis in human adrenal cortex-derived NCI H295R cells. For this purpose a panel of 10 steroids was measured by liquid chromatographic-tandem mass spectrometry. Time-dependent changes in cell incubate concentrations of steroids – including cortisol, aldosterone, dehydroepiandrosterone and their precursors – were measured after incubation with angiotensin II, forskolin and abiraterone. Model parameters were estimated based on experimental data using weighted least square fitting. Time-dependent angiotensin II- and forskolin-induced changes were observed for incubate concentrations of precursor steroids with peaks that preceded maximal increases in aldosterone and cortisol. Inhibition of 17-alpha-hydroxylase/17,20-lyase with abiraterone resulted in increases in upstream precursor steroids and decreases in downstream products. Derived model parameters, including rate constants of enzymatic processes, appropriately quantified observed and expected changes in metabolic pathways at multiple conversion steps. Our data demonstrate limitations of single time point measurements and the importance of assessing pathway dynamics in studies of adrenal cortical cell line steroidogenesis. Our analysis provides a framework for evaluation of steroidogenesis in adrenal cortical cell culture systems and demonstrates that computational modeling-derived estimates of kinetic parameters are an effective tool for describing perturbations in associated metabolic pathways.

DHEA Administration Activates Transcription of Muscular Lipid Metabolic Enzymes via PPARα and PPARδ in Obese Rats.

Administration of dehydroepiandrosterone (DHEA), a precursor of sex steroid hormones, reduces total and visceral fat mass and elevates adipocytic adiponectin gene expression. The aim of this study is to investigate whether levels of peroxisome proliferator-activated receptors (PPARs) in muscle and transcription of PPAR target genes are affected by long-term DHEA administration or exercise training, and whether altered PPAR levels are associated with circulating adiponectin level in obese rats. After 14 weeks on a high-sucrose diet, obese male Wistar rats were assigned randomly to one of 3 groups: control, DHEA administration (1 mg/kg body weight), or exercise training (treadmill running for 1 h, 25 m/min, 5 days/week) for 6 weeks (n=7 for each group). Plasma DHEA and total adiponectin levels in the DHEA-treated and exercise-training groups were significantly higher than those in the obese control group. Additionally, DHEA administration and exercise training significantly increased muscular PPARα and PPARδ protein levels, with a concomitant increase in mRNA expression of 3β-hydroxyacyl-CoA dehydrogenase and cytochrome c oxidase IV, which are target genes of PPARα and PPARδ respectively. Moreover, DHEA administration increased these protein and mRNA levels to the same degree as exercise training. Circulating adiponectin level was positively correlated with plasma DHEA and with muscle levels of PPARα and PPARδ. These results suggest that in obese rats, secretion of adiponectin due to chronic DHEA administration and exercise training may contribute to an increase in the transcription of genes encoding lipid metabolic enzymes, mediated via elevated expression of PPARα and PPARδ in muscle.

Abstract 5422: Inhibition of oxidosqualene cyclase blocks proliferation and survival of prostate cancer cells

Abstract Despite concerted efforts to develop new strategies for preventing and treating prostate cancer, almost 240,000 new cases of the disease are reported each year in the United States, and more than 28,000 men die of the disease annually. Most deaths occur due to complications following metastasis from the primary prostate tumor to other tissues and organs, a process dependent upon increased angiogenesis. Human prostate cancer cells are often dependent on endogenous or exogenous androgens and estrogens, which stimulate cell proliferation and metastasis and inhibit cell death. Standard treatment for primary prostate cancer often includes systemic exposure to chemotherapeutic drugs or antihormones (chemical castration); however, drug-resistant cancer cells often emerge during treatment, limiting the continued use of systemic chemotherapy. Therefore, novel, less toxic and more effective treatments for prostate cancer are urgently needed. The cholesterol biosynthetic pathway is an attractive therapeutic target for endocrine dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as being the metabolic precursor of endogenous steroid hormones. Due to certain undesirable side-effects associated with statins, such as reduced levels of isoprenoids, defective post-translational modification of membrane proteins, and impaired membrane structure and function, alternative approaches are under consideration. 2, 3-oxidosqualene cyclase (OSC) is a key enzyme in the cholesterol biosynthetic pathway downstream of HMGCoA-reductase, the target of statins. While studying anti-cancer properties of Ro 48-8071 [4′-[6-(Allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate]) (RO), an OSC inhibitor, we found that as well as reducing tumor cell viability, RO simultaneously induced the anti-proliferative protein estrogen receptor β (ERβ). Moreover, RO also elevated ERβ in hormone-insensitive cells that are difficult to treat clinically. Initial studies suggest that combining RO with an ERβ agonist increases the antiproliferative effects of RO in prostate cancer cells. RO also induced apoptosis in prostate cancer cells, determined by FACS analysis. In addition, RO effectively reduced the growth of prostate cancer xenografts (PC3 cells) in vivo without any signs of toxicity to experimental animals. Importantly, RO did not reduce the viability of normal prostate cells (RWPE-1). Our study is the first to demonstrate that disrupting cholesterol biosynthesis through OSC inhibition is a novel and potent means by which to destroy human prostate cancer cells. Further studies are necessary to determine the mechanism of RO-mediated loss of prostate cancer cell viability. Support: Department of Defense Prostate Cancer Pgm grant W81XWH-14-1-0246, NIH grant R21 GM088517, and by a COR grant from the University of Missouri, Columbia. Note: This abstract was not presented at the meeting. Citation Format: Yayun Liang, Benford Mafuvadze, Xiaoqin Zou, Cynthia Besch-Williford, Salman M. Hyder. Inhibition of oxidosqualene cyclase blocks proliferation and survival of prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5422. doi:10.1158/1538-7445.AM2015-5422