Androgen Synthesis Research Articles

Abstract C034: Understanding the role of PARP activity in prostate cancer

Abstract Prostate cancer (PCa) is the second most fatal cancer in men in the United States. African American men have an incidence and death rate two times higher than that of European American men. The androgen receptor (AR) is the primary pro-oncogenic transcription factor responsible for PCa emergence and progression. AR directed therapies including androgen deprivation (ADT), direct receptor antagonism and inhibition of androgen synthesis are the current standard of care for advanced PCa. Adaptive resistance to anti-androgen therapy can occur resulting in castration-resistant prostate cancer (mCRPC). mCRPC remains universally fatal with no curative treatment options, which highlights the need for new treatment regimes. Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors are approved to treat mCRPC tumors that have homologous recombination (HR) mutations. PARP-1 is an enzyme that recruits DNA damage repair enzymes. PARP-1 enzymatic activity is elevated in mCRPC patient tumors compared to primary tumors. PARP1 enzymatic activity has been implicated in driving pro-survival mechanisms through recruitment of DNA-damage repair enzymes and regulation of oncogenic transcription factors, including AR. Although increased PARP activity has been associated with driving cancer cell survival, the mechanisms and consequences of elevated PARP activity in PCa remains an area to be further explored. The biologically relevant doses of five PARPi were established in regular and steroid depleted growth conditions in CRPC models. PARP enzymatic activity was assessed by immunoblotting for changes in PARlyation, revealing that PARP protein remains stable, while enzymatic activity is attenuated upon challenge with PARPi. Transcriptomic analysis will assess changes in gene expression programs that are associated with inhibition of PARP enzymatic activity as it relates to key oncogenic pathways in PCa. It will be imperative to gain a deeper understanding of how PARP-1 manipulation modulates pro-oncogenic transcriptional networks to better inform clinical response. Patient derived explants (PDEs) from patients with distinct racial backgrounds will be used to examine differential PARPi responses as it relates to race. This will serve to unravel the mechanisms attributed to the increased lethality of PCa observed in African American populations. Collectively, these studies and future studies for this project will further establish the impact of PARP on PCa gene expression programs and provide resources for future studies exploring PARP in PCa. Further, they may help elucidate mechanisms to address PCa racial disparities. Citation Format: Moriah Cunningham, Latese Evans, Salome Tchotorlishvili, Jasibel Vasquez Gonzalez, Candice Bizzaro, Tessa Mulders, Matthew Schiewer. Understanding the role of PARP activity in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C034.

Morphohistometric and steroidogenic parameters during testicular and epididymal differentiation in cavy (Galea spixii) fetuses

Sexual differentiation and steroidogenic mechanisms have an important impact on postnatal gonadal phenotypic development. Thus, establishing the activities that lead to male phenotypic development can provide a better understanding of this process. This study examined the prenatal development of cavies to establish morphological and histometric development patterns and protein and enzyme immunolocalization processes that are responsible for androgen synthesis in the testes and epididymis. Histological and histometric analyses of the diameter of the seminiferous cords and epididymal ducts of male fetuses on Days 25, 30, 40, and 50 were performed, as well as immunohistochemistry of the steroidogenic enzymes 5α-reductase and 17β-HSD, the androgen receptor, and the anti-Müllerian hormone (AMH). Our findings showed a cellular grouping of gonocytes from Day 30 onward that was characteristic of the seminiferous cord, which was not present in the lumen at any of the studied dates. From Day 50 onward, the differentiation of the three anatomical regions of the epididymis was evident, the head (caput), body (corpus), and tail (cauda), with tissue distinctions. Furthermore, the diameters of the seminiferous cords and epididymal ducts significantly increased with age. On Day 50, the tail showed the greatest diameter of the three regions. The Sertoli and Leydig cells exhibited AMH immunoreactivity at all dates. In addition, the Leydig cells and epididymal epithelial tissue were immunopositive for 5α-reductase, 17β-HSD, and the androgen receptor; therefore, these factors influenced the development and maintenance of the testis and epididymis during cavy prenatal development.

Growth and Treatment in Congenital Adrenal Hyperplasia: An Observational Study from Diagnosis to Final Height

Introduction: Congenital adrenal hyperplasia (CAH) due to 21α-hydroxylase deficiency results in inadequate cortisol and aldosterone synthesis and concomitant overproduction of adrenal androgens. Despite adequate replacement, impaired growth and overweight remain a clinical challenge. The main objective was to investigate the differences in growth, final height (FH), and body mass index (BMI) between different CYP21A2 genotype groups and glucocorticoid treatment strategies during the different phases of growth. Methods: This is a population-based observational cohort study from diagnosis to FH. A total of 86 subjects were diagnosed with CAH in Sweden during 1989–1994. Eighty subjects were followed until FH. There were no interventions apart from the clinical standard of care treatment for CAH. The main outcome measure was the corrected FH standard deviation score (cFH SDS) and its correlation with genotype, accumulated total glucocorticoid dose, and treatment strategy. In addition, BMI and growth trajectories during infancy, childhood, and adolescence were studied. Results: FH was shorter in patients with the more severe CYP21A2 genotypes. Treatment doses of glucocorticoid were within the international treatment recommendations (10–15 mg/m2). Patients with the null and I2 splice genotypes lost approximately 1 SD in FH, whereas patients with the milder genotypes (I172N, P30L, and V281L) were within 0.5 to 0 SDS from target height. cFH SDS was negatively affected by the use of prednisolone but did not correlate with overall glucocorticoid treatment dose calculated as hydrocortisone equivalents. BMI at 18 years was higher in patients treated with prednisolone but did not correlate with genotype. Conclusions: Corrected FH was more affected in patients with severe CYP21A2 genotypes. The addition of a low dose of prednisolone to the hydrocortisone treatment, despite an equivalent total dose of glucocorticoids, was associated with shorter FH and higher BMI in growing subjects with CAH.

In vitro antiandrogenic effects of the herbicide linuron and its metabolites

Although the herbicide linuron is banned for use in the EU due to its reproductive and developmental toxicity, it can still be found in randomly sampled foods grown in and outside the EU. It is not clear if metabolites of linuron can contribute to the endocrine disrupting effects following exposure to the parent compound. To address this gap, we analysed linuron and the metabolites 1-(3,4-dichlorophenyl) urea (DCU), 3,4-dichloroaniline (DCA) and 1-(3,4-dichlorophenyl)-3-methoxyurea (DCXU) for androgen receptor (AR) activities and effects on steroidogenesis. Generally, linuron and the metabolites showed qualitatively similar antiandrogenic profiles, but potencies varied. All compounds were AR antagonists, with linuron showing highest potency (IC50 of 2.8 μM). The overall picture of effects on steroidogenesis showed that linuron and metabolites increased the levels of estrogens and corticosteroids, whereas the synthesis of androgens was inhibited. The metabolite DCU was by far the most potent inhibitor of testosterone synthesis (IC50 of 6.7 μM compared to IC50 of 51.1 μM for linuron). We suggest that it is likely that the metabolites contribute to the antiandrogenic effects of linuron in vivo, especially by inhibiting testosterone synthesis.

A common germline variant in CYP11B1 is associated with adverse clinical outcome of treatment with abiraterone or enzalutamide

Extragonadal androgens play a pivotal role in prostate cancer disease progression on androgen receptor signaling inhibitors (ARSi), including abiraterone and enzalutamide. We aimed to investigate if germline variants in genes involved in extragonadal androgen synthesis contribute to resistance to ARSi and may predict clinical outcomes on ARSi. We included ARSi naive metastatic prostate cancer patients treated with abiraterone or enzalutamide and determined 18 germline variants in six genes involved in extragonadal androgen synthesis. Variants were tested in univariate and multivariable analysis for the relation with overall survival (OS) and time to progression (TTP) by Cox regression, and PSA response by logistic regression. A total of 275 patients were included. From the investigated genes CYP17A1, HSD3B1, CYP11B1, AKR1C3, SRD5A1 and SRD5A2, only rs4736349 in CYP11B1 in homozygous form (TT), present in 54 patients (20%), was related with a significantly worse OS (HR = 1.71, 95% CI 1.09 – 2.68, p = 0.019) and TTP (HR = 1.50, 95% CI 1.08 – 2.09, p = 0.016), and was related with a significantly less frequent PSA response (OR = 0.48, 95% CI 0.24 – 0.96, p = 0.038) on abiraterone or enzalutamide in a multivariable analysis. The frequent germline variant rs4736349 in CYP11B1 is, as homozygote, an independent negative prognostic factor for treatment with abiraterone or enzalutamide in ARSi naive metastatic prostate cancer patients. Our findings warrant prospective investigation of this potentially important predictive biomarker.

Metastatic castration-resistant prostate cancer-emerging trends in therapy

An increasing understanding of the cellular processes involved in growth, metastasis and development of resistance enable the development of new treatment strategies for advanced prostate cancer. Using selected examples, the aim of this report is to present current developments to the reader and to give an outlook on possible upcoming changes in the treatment of advanced prostate cancer. Narrative report based on expert consensus, supported by aliterature search in PubMed (MEDLINE) and the abstract databases of the American Society of Clinical Oncology (ASCO) and European Society of Medical Oncology (ESMO). Examples were selected to illustrate current developments without claiming completeness. The androgen receptor (AR) signal transduction pathway remains afocus of scientific interest. Androgen synthesis inhibitors and AR degraders are promising new approaches to overcome resistance mediated by AR mutations or splice variants. Inhibition of key switch sites of alternative signaling pathways such as AKT or CDK4/6 provide additional treatment options, including combinational strategies through atight linkage with the AR signaling pathway. Abetter understanding of tumor microenvironment and immune response is required for novel immunotherapeutic strategies using bispecific T‑cell engagers (BiTEs) and chimeric antigen receptor (CAR) Tcells. New treatment strategies give hope that we will be able to intervene even more effectively in the course of disease of advanced prostate cancer in the future. However, amajor challenge, especially for the implementation of targeted treatment approaches, is likely to be the heterogeneity of tumor progression not only inter- but also intrapersonally.

Semaphorin 3C promotes de novo steroidogenesis in prostate cancer cells.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

SAT383 Case Study: Impact of Weight Reduction and Oral Contraceptive Pills on Hyperandrogenism

Abstract Disclosure: A. Syeda: None. T.L. Anderson: None. D. Millar: None. P. Madhavan: None. Introduction: Polycystic ovarian syndrome (PCOS) is a commonly encountered cause of hyperandrogenism which could interfere with fertility. Typical therapeutic options for hyperandrogenism including hormonal contraceptives and anti-androgen medication are contraindicated when planning pregnancy. This case highlights the profound impact of weightloss and oral contraception use to treat severe hyperandrogenism in select cases. Case: A 31-year-old female presented for initial evaluation of hyperandrogenism noted during evaluation for infertility. Laboratory investigations revealed a free testosterone level of 172 ng/dL (n:9-55 ng/dl), SHBG of 17 nmol/L (25-122 nmol/L), DHEAS of 306 ug/dL (n:99-340 ug/dL), TSH of 1.79 uIU/mL (n:0.35-4.96 uIU/mL), ACTH of 12.1 (n:7.2-63.3 pg/mL), cortisol of 9.6 ug/dL (7-23 ug/dL), prolactin of 7.85 ng/mL (3.34-26.72 ng/mL). The patient reported hirsutism, increasing body hair, darkening of skin and significant weight gain over the past 4 years. Menstrual cycles have been reported as being irregular since menarche. Vital signs were stable with a blood pressure of 112/84 mm Hg. Physical examination was remarkable for generalized obesity, hirsutism, and acanthosis nigricans, no abnormal abdominal striae were appreciated. Considering significant elevation in testosterone levels despite low SHBG, an MRI of abdomen and pelvis was obtained which demonstrated ovarian cysts with no concern for ovarian malignancy. CT of the abdomen without contrast was negative for adrenal mass. The overall clinical picture was suggestive of a diagnosis of PCOS. As the patient was planning pregnancy, oral contraceptive agents and spironolactone were deferred initially. The patient was encouraged to lose weight with lifestyle modifications and was started on up titrating dose of Metformin as tolerated. The patient complied with the recommendations and over the course of two years her weight decreased from 171 lbs. to 158 lbs. In the interim, she was placed on oral contraceptive pills for 2 months to undergo hysteroscopy. Subsequently, the testosterone levels normalized to 17 ng/dL. Conclusion: Although previous research has demonstrated the significance of weight loss, for reducing symptoms in patients with PCOS, this role has not been well elucidated in cases of severe hyperandrogenism without a neoplastic cause. The mechanism of interaction between weight loss, oral contraceptive pills and decreased androgens is based on the reduction in insulin resistance which causes a decrease in theca cell androgen synthesis and an increased SHBG. Additionally, a decrease in adiposity reduces the conversion of Δ4-androstenedione to testosterone. Notably, in this case there is potentially a causal relationship between the patient's weight loss of 13 pounds (7.6% of her body weight), OCP use and her return to normal testosterone levels. Presentation Date: Saturday, June 17, 2023

FRI376 Regulation Of Theca Cell Function By Salt-Inducible Kinases

Abstract Disclosure: M. Rodriguez Esquivel: None. E. Hayes: None. O. Lakomy: None. M. Hassan: None. R. Filzen: None. C.O. Stocco: Grant Recipient; Self; NIH R01HD097202. The most common identifiable factors of female infertility are ovulatory disorders. Ovulation is the pinnacle of folliculogenesis, a process that requires an interplay between the oocyte, the granulosa cells, and the theca cells (TCs). TCs are the only source of ovarian androgens. Androgens play vital roles in female fertility acting as substrates for estrogen production in granulosa cells and directly regulating ovarian function via the activation of the androgen receptor. However, abnormally elevated androgen levels reduce fertility in humans and animals, suggesting that a delicate equilibrium exists between the beneficial and harmful effects of androgens in the ovary. Therefore, uncovering novel mechanisms regulating androgen synthesis in TCs is of great significance. The capacity of TCs to synthesize androgens relies on the expression of the rate-limiting enzyme CYP17A1, which converts progestogens into androgens, and its stimulation by the luteinizing hormone (LH). Previously, our group showed that salt-inducible kinases (SIKs) regulate granulosa cell steroidogenesis. Here, we investigated whether SIKs play any role in the regulation of androgen production in TCs. SIK2 and SIK3 were detected by IHC in the TCs of rat ovaries. Western blot confirmed this finding using isolated rat TCs. Next, rat TCs in culture were treated with LH in the presence or absence of HG, a highly specific SIK inhibitor. SIK inhibition enhanced the stimulatory effect of LH on Cyp17a1 mRNA expression. HG alone also increased Cyp17a1 expression to levels higher than those found in the presence of LH. Similarly, HG alone increased testosterone and potentiated LH stimulation of testosterone production. SIK inhibition also enhanced the stimulatory effect of LH on the expression of StAR and Cyp11a1. Concentration-response experiments for the SIK inhibitor in the presence or absence of LH show that SIK inhibition stimulates Cyp17a1 in a concentration-dependent manner from 0.1 to 1 µM. Of note, at 3 µM, HG was less effective in inducing Cyp17a1 than at 1 µM, suggesting that higher concentrations of HG may have alternative unknown effects. Activation of adenyl cyclase with forskolin or emulation of increased intracellular cAMP with dbcAMP stimulated Cyp17a1 an effect that was also enhanced by the inhibition of SIK. Since the main target of cAMP is PKA, we used lentivirus to express constitutively active PKA (caPKA). Overexpression of caPKA was sufficient to stimulate testosterone production, an effect that was significantly augmented by SIK inhibition, suggesting that SIKs regulate factors downstream of PKA. This evidence shows that TCs express SIKs and reveal novel roles for SIKs in the regulation of TC function and androgen production. This information could contribute to uncovering therapeutic targets to treat hyperandrogenic diseases such as PCOS. Presentation: Friday, June 16, 2023

Adverse effect of environmental androgenic compounds Galaxolide and Irgacure 369 on the male reproductive system

Our recent study has found that two environmental chemicals, Galaxolide (HHCB, a raw material for synthesizing musk) and Irgacure 369 (IC-369, a photoinitiator used in packaging) are agonists for the androgen receptor in vitro and in vivo. This study aims to reveal the subchronic reproductive toxicity of these two compounds in mature male rats. The results showed that compared with the control group, HHCB and IC-369 reduced the sperm concentration and motility, increased the sperm deformity, and caused the atrophy of the seminiferous tubules in the testicles. Exposure to HHCB and IC-369 reduced testosterone level, and induced luteinizing hormone, and follicle-stimulating hormone levels in rat serum. Compared with the control group, the levels of oxidative stress markers in the serum and testicular tissue increased. HHCB and IC-369 also inhibited expression of the genes involved in androgen synthesis in testicle. The above results indicated that HHCB and IC-369 could affect the levels of sex hormones, alter gene expression profiles and induce histological damage in reproductive organs, resulting in decreased sperm quality. Therefore, HHCB and IC-369 have endocrine disruptors with prominent reproductive toxicity in males.

Predictors of surgical complications in boys with hypospadias: data from an international registry

BackgroundComplications are frequently reported after hypospadias repair and there is a need to understand the factors that influence their occurrence.MethodsData from boys with hypospadias born between 2000 and 2020 were...

Gynecological and dermatological aspects of diagnostics of polycystic ovary syndrome from puberty to menopause

The article is devoted to the review of scientific publications on gynecological and dermatological aspects of polycystic ovary syndrome (PCOS) in different age periods of women’s life. Analysis of domestic and foreign publications presents that the prevalence of PCOS depends on the age of women and the state of their reproductive function, and is accounted 17% in women 21 to 30 years old and significantly decreased with age. More than half of all cases of endocrine infertility (50-75%) and about 20-22% of the causes of infertile marriage in general are associated with PCOS. The phenotypic heterogeneity of women with PCOS affects the pregnancy outcomes in different ways, which increases the risk of its pathological course, early pregnancy loss, the development of gestational diabetes mellitus, hypertensive disorders, the birth of small and large for gestational age babies, etc.“The golden standard” for hyperandrogenism diagnosis in PCOS patients is the determination of the index of free testosterone and androstenedione. Other indicators are important for differential diagnosis and definition of syndrome phenotypes. Different PCOS phenotypes are characterized by different ratios of the contribution of sources of excess androgen synthesis. Depending on the cause, hyperandrogenism in PCOS patients is accompanied by various metabolic risks. Usually, an excess of androgens in women is clinically manifested by hirsutism, seborrheic dermatitis, acne, acanthosis nigricans, and androgenetic alopecia.The presence of dermatological manifestations of hyperandrogenism has different diagnostic value in puberty, women of reproductive age and in menopause. Acne can be the first sign of pubertal maturation. Additional examinations should be planned in extremely severe cases, which are accompanied by signs of androgen excess, or do not respond to treatment. In women of reproductive age and in perimenopause, the need to assess androgenic status is extremely important. Hair loss according to the female type is associated with manifestations of metabolic syndrome, and it is an independent risk factor for the development of diabetes, atherosclerosis and mortality from cardiovascular diseases.Therefore it is recommended to use the modified Rotterdam criteria for PCOS diagnosis. Such criteria include clinical or biochemical hyperandrogenism, signs of oligoanovulation, polycystosis (morphology of the ovaries according to ultrasound diagnostics), if other causes of relevant disorders are excluded. At the same time, any two of the specified criteria have diagnostic value, which makes possible to establish not only the diagnosis, but also clinical variant (phenotype) of PCOS, the diagnosis of which is the basis for choosing individual treatment for this contingent of women.

Association between immunity and different clinical symptoms in patients with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a disease with endocrine and metabolic disorders. The main symptoms are hyperandrogenemia (HA), insulin resistance (IR), and ovulation disorder. However, the pathogenesis and pathophysiological process of these major symptoms in PCOS are still not well defined. In recent studies, the chronic low-grade inflammatory state has become one of the factors affecting PCOS. Some alterable immune factors in PCOS, such as interleukin-15 and interleukin-1, have been identified to be related to androgen synthesis and insulin resistance in PCOS. In addition, a disturbed immune microenvironment in the ovary leads to impaired follicular growth and ovulation. Previous studies have roughly reviewed the relationship between immunity and PCOS. However, the link between the different clinical manifestations of PCOS and immunity has not been well explored and analyzed. The clinical presentation of each patient is diverse, and symptomatic treatment is mainly used. Therefore, this article reviews several representative immunological factors that affect these three symptoms to explore the underlying mechanism, which will be beneficial for developing new treatment strategies.

Changes of androgen and corticosterone metabolites excretion and conversion in cystic fibrosis.

Cystic fibrosis (CF) is a life-threatening inherited disease related to a mutation in the CFTR gene, that leads to serious health complications such as chronic pulmonary infections, pancreatic insufficiency, dysfunction of the sweat glands and reproductive system. For the first time, we have described the profile of corticosterone and androgen metabolites in urine, as well as the activity of enzymes involved in steroid genesis and metabolism in people with CF, using gas chromatography/mass spectrometry. A significant reduction in the excretion of most of the measured metabolites in CF was found. These differences were observed in the group of progestagen metabolites, as well as among metabolites of corticosterone and androgens. We revealed higher activities of 17β-hydroxysteroid dehydrogenase and 17,20-lyase in the Δ4 pathway compared with controls, what can promote the androgen synthesis through the backdoor androgen pathway. We have also found the increased conversion activity of 11-oxyganated steroids by 5a-reductase in backdoor pathway. Levels of the most potent and vital androgens (testosterone and dihydrotestosterone) are comparable in both groups. However, the excretion of dehydroepiandrosterone was lower in CF. Decreased cholesterol lipoprotein levels may contribute to limited intracellular cholesterol supply and reduced adrenal steroidogenesis in CF individuals. Changes in the activity of some steroidogenesis enzymes may suggest the presence of some peripheral adaptive mechanisms in CF to maintain androgen balance in the body despite the limited sufficiency of secretion by the adrenal cortex.

CYP11A1 silencing suppresses HMGCR expression via cholesterol accumulation and sensitizes CRPC cell line DU-145 to atorvastatin

Statins, which are cholesterol synthesis inhibitors, are well-known therapeutics for dyslipidemia; however, some studies have anticipated their use as anticancer agents. However, epithelial cancer cells show strong resistance to statins through an increased expression of HMG-CoA reductase (HMGCR), an inhibitory target of statins. Castration-resistant prostate cancer (CRPC) cells synthesize androgens from cholesterol on their own. We performed suppression of CYP11A1, a rate-limiting enzyme in androgen synthesis from cholesterol, using siRNA or inhibitors, to examine the effect of steroidogenesis inhibition on statin sensitivity in CRPC cells. Here, we suggested that CYP11A1 silencing sensitized the statin-resistant CRPC cell line DU-145 to atorvastatin via HMGCR downregulation by an increase in intracellular free cholesterol. We further demonstrated that CYP11A1 silencing induced epithelial-mesenchymal transition, which converted DU-145 cells into a statin-sensitive phenotype. This suggests that concomitant use of CYP11A1 inhibitors could be an effective approach for overcoming statin resistance in CRPC. Moreover, we showed that ketoconazole, a CYP11A1 inhibitor, sensitized DU-145 cells to atorvastatin, although not all the molecular events observed in CYP11A1 silencing were reproducible. Although further studies are necessary to clarify the detailed mechanisms, ketoconazole may be effective as a concomitant drug that potentiates the anticancer effect of atorvastatin.

Synthesis of 6β-hydroxy androgens from a 3,5-diene steroid precursor to test for cytochrome P450 3A4-catalyzed hydroxylation of androstenedione

6β-Hydroxytestosterone is a biomarker for the activity of human cytochrome P450 3A4 (P450 3A4), the major drug metabolizing cytochrome P450 enzyme. Despite its significance, efficient routes for the chemical synthesis of 6β-hydroxytestosterone are rare. In this study, 6β-hydroxytestosterone was synthesized through the oxidation of a 3,5-diene precursor under the Uemura-Doyle reaction conditions using a dirhodium catalyst in the presence of tert-butylhydroperoxide. Mechanistic studies showed that some oxygen is incorporated from molecular oxygen and CH abstraction is partially rate-limiting. This reaction was used to synthesize 6β-hydroxyandrostenedione, which was used as a standard to test the hypothesis of whether P450 3A4 catalyzes the hydroxylation of androstenedione. Upon incubation of P450 3A4 with androstenedione, a hydroxylated product was formed, which matched the retention time of synthetic 6β-hydroxyandrostenedione. This reaction can be exploited to study other biochemical processes involving compounds with a 6 β -hydroxy-3-keto-Δ4 steroid backbone.

CircEpha5 regulates the synthesis and secretion of androgen in mouse preantral follicles by targeting miR-758-5p

Circular RNAs are involved in the pathogenesis of various diseases, although its expression pattern and role in polycystic ovary syndrome (PCOS), characterised by hyperandrogenism, are not very clear. This article assessed the circRNAs expression profile in the ovaries of PCOS mice by circRNAs high-throughput sequencing and explored the role of circEpha5 in hyperandrogenism. The results showed that the overexpression of circEpha5 in mouse preantral follicles could increase the expression of Cyp17a1, an androgen synthesis-related gene, which resulted in a higher serum level of testosterone. Dual-luciferase reporter gene studies identified miR-758-5p as a direct target of circEpha5. Consequently, miR-758-5p expression was downregulated upon circEpha5 overexpression. Ectopically expressed miR-758-5p reversed the stimulation effects of circEpha5 on steroidogenesis-related gene expression and testosterone release. Therefore, circEpha5 could sponge miR-758-5p to regulate the expression of Cyp17a1, thereby promoting the synthesis and secretion of androgen in the preantral follicles. This work is contributed to the understanding of the pathogenesis of hyperandrogenemia and lays the foundation for the development of therapeutic targets of PCOS hyperandrogenism.

Adrenal steroid metabolites and bone status in patients with adrenal incidentalomas and hypercortisolism

Autonomous cortisol secretion (ACS), resulting from cortisol-producing adenomas (CPA), causes endogenous steroid-induced osteoporosis (SIOP). However, the risk of endogenous SIOP cannot be explained by cortisol excess alone, and how other steroid metabolites affect bone status is unclear. ACS was diagnosed as serum cortisol ≥1.8μg/dL after the 1-mg dexamethasone suppression test (DST-cortisol). Using liquid chromatography tandem mass spectrometry, 21 plasma steroid metabolites were measured in 73 patients with ACS and 85 patients with non-functioning adrenal tumors (NFAT). Expression of steroidogenic enzymes and relevant steroid metabolites were analyzed in some of CPA tissues. Discriminant and principal component analyses distinguished steroid profiles between the ACS and NFAT groups in premenopausal women. Premenopausal women with ACS exhibited higher levels of a mineralocorticoid metabolite, 11-deoxycorticosterone (11-DOC), and lower levels of androgen metabolites, dehydroepiandrosterone-sulfate, and androsterone-glucuronide. In premenopausal women with ACS, DST-cortisol negatively correlated with trabecular bone score (TBS). Additionally, 11-DOC negatively correlated with lumbar spine-bone mineral density, whereas androsterone-glucuronide positively correlated with TBS. The CPA tissues showed increased 11-DOC levels with increased expression of CYP21A2, essential for 11-DOC synthesis. Adrenal non-tumor tissues were atrophied with reduced expression of CYB5A, required for androgen synthesis. This study demonstrates that unbalanced production of adrenal steroid metabolites, derived from both adrenal tumor and non-tumor tissues, contributes to the pathogenesis of endogenous SIOP in premenopausal women with ACS. JSPS KAKENHI, Secom Science and Technology Foundation, Takeda Science Foundation, Japan Foundation for Applied Enzymology, AMED-CREST, JSTA-STEP, JST-Moonshot, and Ono Medical Research Foundation.

Salt-inducible kinases regulate androgen synthesis in theca cells by enhancing CREB signaling

Ovulation is the pinnacle of folliculogenesis, a process that requires an interplay between the oocyte, the granulosa cells, and the theca cells (TCs). TCs are the only source of ovarian androgens, which play a vital role in female fertility. However, abnormally elevated androgen levels reduce fertility. Therefore, uncovering novel mechanisms regulating androgen synthesis in TCs is of great significance. We have shown that salt-inducible kinases (SIKs) regulate granulosa cell steroidogenesis. Here, we investigated whether SIKs regulate androgen production in TCs. SIK2 and SIK3 were detected in the TCs of mouse ovaries and isolated TCs. Next, TCs in culture were treated with luteinizing hormone (LH) in the presence or absence of a highly specific SIK inhibitor. SIK inhibition enhanced the stimulatory effect of LH on steroidogenic gene expression and androgen production in a concentration-dependent manner. SIK inhibition alone stimulated the expression of steroidogenic genes and increased androgen production. Activation of adenylyl cyclase with forskolin or emulation of increased intracellular cyclic AMP levels stimulated steroidogenesis, an effect that was enhanced by the inhibition of SIK activity. The stimulatory effect of downstream targets of cyclic AMP was also significantly augmented by SIK inhibition, suggesting that SIKs control targets downstream cyclic AMP. Finally, it is shown that SIK2 knockout mice have higher circulating testosterone than controls. This evidence shows that TCs express SIKs and reveal novel roles for SIKs in the regulation of TC function and androgen production. This information could contribute to uncovering therapeutic targets to treat hyperandrogenic diseases.

Discovery of a Novel Bifunctional Steroid Analog, YXG-158, as an Androgen Receptor Degrader and CYP17A1 Inhibitor for the Treatment of Enzalutamide-Resistant Prostate Cancer.

The androgen/androgen receptor (AR) signaling pathway plays an important role in castration-resistant prostate cancer (CRPC). Bifunctional agents that simultaneously degrade AR and inhibit androgen synthesis are expected to block the androgen/AR signaling pathway more thoroughly, demonstrating the promising therapeutic potential for CRPC, even enzalutamide-resistant CRPC. Herein, a series of steroid analogs were designed, synthesized, and identified as selective AR degraders, among which YXG-158 (23-h) was the most potent antitumor compound with dual functions of AR degradation and CYP17A1 inhibition. In addition, 23-h abrogated the hERG inhibition and exhibited excellent PK profiles. In vivo, 23-h effectively inhibited the growth of hormone-sensitive organs in the Hershberger assay and exhibited robust antitumor efficacy both in enzalutamide-sensitive (LNCaP/AR) and enzalutamide-resistant (C4-2b-ENZ) xenograft models. Thus, 23-h was chosen as a preclinical candidate for the treatment of enzalutamide-resistant prostate cancer.