Steroid Precursors Research Articles

Prostate cancer androgen biosynthesis relies solely on CYP17A1 downstream metabolites

Prostate cancer (PC) is dependent on androgen receptor (AR) activation by testosterone and 5α-dihydrotestosterone (DHT). Intratumoral androgen accumulation and activation despite systemic androgen deprivation therapy underlies the development of castration-resistant PC (CRPC), but the precise pathways involved remain controversial. Here we investigated the differential contributions of de novo androgen biosynthesis and androgen precursor conversion to androgen accumulation. Steroid flux analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on (CR)PC cell lines and fresh patient PC tissue slices after incubation with classic and alternative biosynthesis intermediates, alongside quantitative PCR analysis for steroidogenic enzyme expression. Activity of CYP17A1 was undetectable in all PC cell lines and patient PC tissue slices. Instead, steroid flux analysis confirmed the generation of testosterone and DHT from adrenal precursors and reactivation of androgen metabolites. Precursor steroids upstream of DHEA were converted down the first steps of the alternative DHT biosynthesis pathway, but did not proceed through to active androgen generation. Comprehensive steroid flux analysis of (CR)PC cells provides strong evidence against intratumoral de novo androgen biosynthesis and demonstrates that androgen precursor steroids downstream of CYP17A1 activities constitute the major source of intracrine androgen generation.

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

Draft genome sequence of soil isolate Mycolicibacterium fortuitum DVD-1301

ABSTRACT Some strains of Mycolicibacterium possess high sterol-oxidizing activity and are used in the pharmaceutical industry for the production of steroid precursors. Herein, we report a draft genome sequence of the soil-dwelling Mycolicibacterium fortuitum DVD-1301 isolated in the floodplain of the river Oka. The genome contains a full set of steroid catabolic genes.

Anticancer and anti-metastasis activity of 1,25 dihydroxycholecalciferols and genistein in MCF-7 and MDA-MB-231 breast cancer cell lines

A powerful steroid hormone precursor, 1,25 dihydroxycholecalciferols (1,25(OH)2D3), and dietary phytoestrogen (genistein) are essential compounds that act by binding to nuclear receptors and altering gene expression. They have many biological benefits, some of which have anticancer properties. We studied the impact of 1,25(OH)2D3 and genistein on the proliferation, progression, and metastasis of MCF-7 and MDA-MB-231 cells when they were used alone or in combination and investigated whether there was a synergistic effect between genistein and 1,25(OH)2D3. To achieve these goals, a variety of assays, including flow cytometry, cell invasion assays, cell adhesion assays, Western blotting, and RT‒PCR, were used.Our findings showed that genistein, 1,25(OH)2D3, and the two combined all effectively declined the growth of MCF-7 and MDA-MB-231 cells by arresting the cells in the G0/G1 phase and inducing an apoptotic pathway. Stimulation of apoptosis was achieved by upregulating the expression of BAX and CASP3 genes and downregulating the expression levels of BCL-2 gene. Furthermore, both compounds suppress metastasis by reducing cell adhesion and cell migration/invasion by elevating the expression level of E-cadherin and reducing the expression level of P-cadherin and N-cadherin. Additionally, both genistein and 1,25(OH)2D3 increased the expression level of ERK1 and reduced the expression levels of JNK, p38, Ras, and MEK proteins, which reduced metastasis, enhanced the response to cancer treatment, and improved overall survival. Thus, genistein and 1,25(OH)2D3 can both be considered key candidates in the search for new breast cancer treatments.

Expression of genes and enzymes involved in ovarian steroidogenesis in relation to human follicular development.

Granulosa cells (GCs) and theca cells (TCs) play a pivotal role in human ovarian steroidogenesis, facilitating the conversion of cholesterol into sex steroids that regulate normal reproductive function. This study aims to explore the expression patterns of key enzymes that govern human ovarian steroidogenesis throughout follicle development, employing both genomic and immunological methodologies. Follicles and GCs obtained from women undergoing ovarian tissue cryopreservation (OTC) and in vitro fertilisation treatment were utilized. Gene expression data were obtained from a Chinese study using RNA sequencing and from microarray data generated in our laboratory to comprehensively analyse gene expression profiles across distinct stages of follicular development. To corroborate the localisation of key enzymes within GCs and TCs, immunohistochemistry analyses utilizing colourimetric and fluorescent techniques were conducted. Steroidogenesis-related enzymes displayed low gene expression levels during early follicle development. However, a notable upregulation of HSD3B2 was observed in GCs as follicles progressed to the antral/preovulatory stage, confirmed consistently using both microarray and RNA sequencing methodologies. Furthermore, immunohistochemical analyses effectively demonstrated that HSD3B2 were not only expressed in GCs, but co-localised with CYP17A1 within a specific subset of TCs surrounding human small antral follicles. Contributing to an enhanced progesterone production during the second half of the follicular phase was a significant upregulation of CYB5A in both microarray and RNA-seq datasets as follicles transition from the antral stage to the pre-ovulatory stage. Moreover, an augmented expression of DHCR24 and LDLR in both types of data, along with HMGCR expression expression in the microarray data, indicates increased substrate availability for ovarian steroidogenesis. This study confirms and extends that GCs gradually augment expression of HSD3B2 thereby enhancing their capacity for progesterone synthesis as follicles reach the size of selection at around 10 mm in diameter. This is supported by the expression CYB5A and possibly augmented availability of steroid precursors. A subset of TCs exhibit concurrent expression of CYP17A1 and HSD3B2, collectively contributing to the synthesis of 17-hydroxyprogesterone. These data significantly enhance our understanding of the dynamic regulation of progesterone throughout the process of follicular development.

Medicinal-Chemistry-Driven Approach to 2-Substituted Benzoxazole-Estradiol Chimeras: Synthesis, Anticancer Activity, and Early ADME Profile.

The efficient synthesis of novel estradiol-based A-ring-fused oxazole derivatives, which can be considered as benzoxazole-steroid domain-integrated hybrids containing a common benzene structural motif, is described. The target compounds were prepared from steroidal 2-aminophenol precursors by heterocycle formation or functional group interconversion (FGI) strategies. According to 2D projection-based t-distributed stochastic neighbor embedding (t-SNE), the novel molecules were proved to represent a new chemical space among steroid drugs. They were characterized based on critical physicochemical parameters using in silico and experimental data. The performance of the compounds to inhibit cell proliferation was tested on four human cancer cell lines and non-cancerous cells. Further examinations were performed to reveal IC50 and lipophilic ligand efficiency (LLE) values, cancer cell selectivity, and apoptosis-triggering features. Pharmacological tests and LLE metric revealed that some derivatives, especially the 2-(4-ethylpiperazin-1-yl)oxazole derivative exhibit strong anticancer activity and trigger the apoptosis of cancer cells with relatively low promiscuity risk similarly to the structurally most closely-related and intensively studied anticancer agent, 2-methoxy-estradiol.

VDR promotes testosterone synthesis in mouse Leydig cells via regulation of cholesterol side chain cleavage cytochrome P450 (Cyp11a1) expression.

The vitamin D receptor (VDR) mediates the pleiotropic biological actions that include osteoporosis, immune responses and androgen synthesis.VDR is widely expressed in testis cells such as Leydig cells, Sertoli cells, and sperm. The levels of steroids are critical for sexual development. In the early stage of steroidogenesis, cholesterol is converted to pregnenolone (precursor of most steroid hormones) by cholesterol side-chain lyase (CYP11A1), which eventually synthesizes the male hormone testosterone. This study aims to reveal how VDR regulates CYP11A1 expression and affects testosterone synthesis in murine Leydig cells. The levels of VDR, CYP11A1 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. Targeted relationship between VDR and Cyp11a1 was evaluated by dual-luciferase reporter assay. The levels of testosterone concentrations in cell culture media serum by enzyme-linked immunosorbent assay (ELISA). Phylogenetic and motif analysis showed that the Cyp11a1 family had sequence loss, which may have special biological functions during evolution. The results of promoter prediction showed that vitamin D response element (VDRE) existed in the upstream promoter region of murine Cyp11a1. Dual-luciferase assay confirmed that VDR could bind candidate VDREs in upstream region of Cyp11a1, and enhance gene expression. Tissue distribution and localizatio analysis showed that Cyp11a1 was mainly expressed in testis, and dominantly existed in murine Leydig cells. Furthermore, over-expression VDR and CYP11A1 significantly increased testosterone synthesis in mice Leydig cells. Active vitamin D3 (VD3) and Vdr interference treatment showed that VD3/VDR had a positive regulatory effect on Cyp11a1 expression and testosterone secretion. VDR promotes testosterone synthesis in male mice by up-regulating Cyp11a1 expression, which played an important role for male reproduction.

Long-term health consequences of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency accounts for 95% of all CAH cases and is one of the most common inborn metabolic conditions. The introduction of life-saving glucocorticoid replacement therapy 70 years ago has changed the perception of CAH from a paediatric disorder into a lifelong, chronic condition affecting patients of all age groups. Alongside health problems that can develop during the time of paediatric care, there is an emerging body of evidence suggesting an increased risk of developing co-morbidities during adult life in patients with CAH. The mechanisms that drive the negative long-term outcomes associated with CAH are complex and involve supraphysiological replacement therapies (glucocorticoids and mineralocorticoids), excess adrenal androgens both in the intrauterine and postnatal life, elevated steroid precursors and adrenocorticotropic hormone levels. Alongside a review of mortality outcome, we discuss issues that need to be addressed when caring for the CAH patient including female and male fertility, cardio-metabolic morbidity, bone health and other important long-term outcomes of CAH.

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.

Cholesterol metabolism in T-cell aging: Accomplices or victims.

Aging has a significant impact on the function and metabolism of T cells. Cholesterol, the most important sterol in mammals, is known as the "gold of the body" because it maintains membrane fluidity, rigidity, and signal transduction while also serving as a precursor of oxysterols, bile acids, and steroid hormones. Cholesterol homeostasis is primarily controlled by uptake, biosynthesis, efflux, and regulatory mechanisms. Previous studies have suggested that there are reciprocal interactions between cholesterol metabolism and T lymphocytes. Here, we will summarize the most recent advances in the effects of cholesterol and its derivatives on T-cell aging. We will furthermore discuss interventions that might be used to help older individuals with immune deficiencies or diminishing immune competence.

An iPSC-derived astrocyte model of fragile X syndrome exhibits dysregulated cholesterol homeostasis

Cholesterol is an essential membrane structural component and steroid hormone precursor, and is involved in numerous signaling processes. Astrocytes regulate brain cholesterol homeostasis and they supply cholesterol to the needs of neurons. ATP-binding cassette transporter A1 (ABCA1) is the main cholesterol efflux transporter in astrocytes. Here we show dysregulated cholesterol homeostasis in astrocytes generated from human induced pluripotent stem cells (iPSCs) derived from males with fragile X syndrome (FXS), which is the most common cause of inherited intellectual disability. ABCA1 levels are reduced in FXS human and mouse astrocytes when compared with controls. Accumulation of cholesterol associates with increased desmosterol and polyunsaturated phospholipids in the lipidome of FXS mouse astrocytes. Abnormal astrocytic responses to cytokine exposure together with altered anti-inflammatory and cytokine profiles of human FXS astrocyte secretome suggest contribution of inflammatory factors to altered cholesterol homeostasis. Our results demonstrate changes of astrocytic lipid metabolism, which can critically regulate membrane properties and affect cholesterol transport in FXS astrocytes, providing target for therapy in FXS.

Comprehensive analysis of the clinical and biological significances of cholesterol metabolism in lower-grade gliomas

BackgroundAs a component of membrane lipids and the precursor of oxysterols and steroid hormones, reprogrammed cholesterol metabolism contributes to the initiation and progression of multiple cancers. Thus, we aim to further investigate the significances of cholesterol metabolism in lower-grade gliomas (LGGs).MethodsThe present study included 413 LGG samples from TCGA RNA-seq dataset (training cohort) and 172 LGG samples from CGGA RNA-seq dataset (validation cohort). The cholesterol metabolism-related signature was identified by the LASSO regression model. Bioinformatics analyses were performed to explore the functional roles of this signature in LGGs. Kaplan-Meier and Cox regression analyses were enrolled to estimate prognostic value of the risk signature.ResultsOur findings suggested that cholesterol metabolism was tightly associated clinicopathologic features and genomic alterations of LGGs. Bioinformatics analyses revealed that cholesterol metabolism played a key role in immunosuppression of LGGs, mainly by promoting macrophages polarization and T cell exhaustion. Kaplan-Meier curve and Cox regression analysis showed that cholesterol metabolism was an independent prognostic indicator for LGG patients. To improve the clinical application value of the risk signature, we also constructed a nomogram model to predict the 1-, 3- and 5-year survival of LGG patients.ConclusionThe cholesterol metabolism was powerful prognostic indicator and could serve as a promising target to enhance personalized treatment of LGGs.

Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells

Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.

Single-Cell RNA-Seq Identifies Pathways and Genes Contributing to the Hyperandrogenemia Associated with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by hyperandrogenemia of ovarian thecal cell origin, resulting in anovulation/oligo-ovulation and infertility. Our previous studies established that ovarian theca cells isolated and propagated from ovaries of normal ovulatory women and women with PCOS have distinctive molecular and cellular signatures that underlie the increased androgen biosynthesis in PCOS. To evaluate differences between gene expression in single-cells from passaged cultures of theca cells from ovaries of normal ovulatory women and women with PCOS, we performed single-cell RNA sequencing (scRNA-seq). Results from these studies revealed differentially expressed pathways and genes involved in the acquisition of cholesterol, the precursor of steroid hormones, and steroidogenesis. Bulk RNA-seq and microarray studies confirmed the theca cell differential gene expression profiles. The expression profiles appear to be directed largely by increased levels or activity of the transcription factors SREBF1, which regulates genes involved in cholesterol acquisition (LDLR, LIPA, NPC1, CYP11A1, FDX1, and FDXR), and GATA6, which regulates expression of genes encoding steroidogenic enzymes (CYP17A1) in concert with other differentially expressed transcription factors (SP1, NR5A2). This study provides insights into the molecular mechanisms underlying the hyperandrogenemia associated with PCOS and highlights potential targets for molecular diagnosis and therapeutic intervention.

Sex Hormones associated with Temporomandibular Pain on Palpation in Male Adolescents - Results of the Epidemiologic LIFE Child Study.

This study aimed to investigate whether the sex steroid precursor hormone dehydroepiandrosterone sulfate (DHEA-S), sex hormone-binding globulin (SHBG), and testosterone (TT) are associated with temporomandibular (TM) pain on palpation in male adolescents. Out of the LIFE Child study dataset containing 1,022 children and adolescents aged 10 to 18 years (496 males, 48.5%), we used a subsample of 273 male adolescents (mean age: 13.8 ± 2.3 years) in advanced pubertal development (PD) to analyze the association between hormones and TM pain. The Tanner scale was applied to describe the stage of PD. Pain on palpation of the temporalis and masseter muscles and the TM joints (palpation pain) was assessed using the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD). Serum levels of sex hormones (DHEA-S, SHBG, TT) were determined using standardized laboratory analyses. Free TT was estimated from the ratio between TT and SHBG (free androgen index = FAI). We calculated the risk of perceived positive palpation pain for male participants as a function of hormone levels (DHEA-S, FAI) taking into account age and body mass index (BMI). Among more developed (Tanner stage 4-5) male adolescents, 22.7% (n=62) reported palpation pain in the TM region. In these participants, FAI levels were approximately half that of individuals without such pain (p<0.01). DHEA-S levels were about 30% lower in the pain group (p<0.01). In multivariable regression analyses, the odds ratio (OR) for pain on palpation decreased to 0.75 (95% confidence interval [CI]: 0.57-0.98) per 10 units of FAI level compared to those without pain, after controlling for the effects of age and adjusted BMI. We observed the same effect for this subgroup per unit of DHEA-S serum level (OR=0.71; 95% CI: 0.53-0.94). At subclinical lower levels of serum free TT and DHEA-S, male adolescents are more likely to report pain on standardized palpation of the masticatory muscles and/or TM joints. This finding supports the hypothesis that sex hormones may influence pain reporting.

Abstract IA004: Metabolic mechanisms of prostate cancer progression that feed nuclear receptors

Abstract The androgen axis is central to prostate cancer physiology and therapy. Androgens are gonadal and extragonadal in origin, both of which stimulate the androgen receptor (AR). The use of medical castration (ADT), adrenal androgen ablation (i.e., abiraterone) and potent AR antagonists (i.e., enzalutamide, apalutamide, darolutamide), has yielded complex clinical states of prostate cancer, including disease states that are resistant to multiple forms of hormonal therapy. One of the major questions that arises is whether men can be identified who will benefit from earlier intensive hormonal therapy. Another question is whether alternative hormonal treatment strategies can be used to address resistance to the current generation of hormonal therapies. We propose that identification of steroid metabolic dependencies is part of the answer to both of these questions. Approximately half of men with prostate cancer inherit an activating missense in the first and rate-limiting enzyme that converts extragonadal precursor steroids to dihydrotestosterone (DHT). The “adrenal-permissive” HSD3B1(1245C) allele encodes for a 3β-hydroxysteroid dehydrogenase-1 (3βHSD1) enzyme that increases tumor DHT synthesis from adrenal DHEA. Heterozygous or homozygous HSD3B1(1245C) inheritance confers poor outcomes after ADT. Furthermore, men with HSD3B1(1245C) inheritance and castration-resistant prostate cancer (CRPC) treated with abiraterone or enzalutamide still have poorer outcomes. Together, these data suggest that directly targeting this metabolic lesion is necessary for optimal therapy in these men. We recently discovered that phosphorylation of the 3βHSD1 tyrosine 344 by the BMX kinase is necessary for androgen biosynthesis and CRPC in xenograft models. These findings have led to the launch of the Maverick clinical trial in the Prostate Cancer Clinical Trials Consortium (PCCTC) that combines abiraterone plus BMX inhibition with abivertinib in men with CRPC. In sum, this accumulating body of evidence suggests that steroid metabolic dependencies should be considered as an essential part of cancer metabolism, particularly in the context of developmental therapeutics for prostate cancer. Citation Format: Nima Sharifi. Metabolic mechanisms of prostate cancer progression that feed nuclear receptors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr IA004.

Relationship between androgen biosynthesis linked to 3βHSD1 and resistance to radiotherapy: A germline biomarker for combined androgen blockade with radiation in high-risk prostate cancer.

e17084 Background: Around 50% of men with advanced prostate cancer (PCa) have at least one germline copy of the adrenal-permissive (1245C) HSD3B1 allele. This allele leads to higher levels of the steroid biosynthesis enzyme, 3β-hydroxysteroid dehydrogenase (3βHSD1). Inheriting this allele is linked to worse outcomes in men with advanced PCa. To determine if (1245C) HSD3B1 could be causing early resistance to combined androgen deprivation and radiotherapy in localized PCa, we studied its role in modulating radioresistance. Methods: PCa cell lines were used to investigate the reciprocal effects of 3βHSD1 knockdown and overexpression in intrinsically high and low 3βHSD1 lines, respectively. PCa xenografts were used to confirm the results in vivo. The connection between androgen receptor (AR) expression and elevated DNA Damage Response (DDR) gene expression was validated using transcriptomic data from 680 radical prostatectomy specimens The ability of enzalutamide, a non-steroidal anti-androgen, to restore radiosensitivity in 1245C expressing lines was interrogated in vitro and in vivo. Results: 1245C HSD3B1 expressing PCa lines displayed increased clonogenic survival after ionizing radiation. Effects were dependent on the 3βHSD1 substrate, DHEA, confirming intratumoral androgen metabolism was required for radioresistance. Resistant lines showed enhanced DNA double-strand break (DSB) repair and heightened DDR gene expression. PCa xenografts with 1245C HSD3B1 were similarly more resistant to radiation using murine models that faithfully mimic human adrenal physiology. A correlation between AR expression and increased DDR gene expression was confirmed in 680 patient samples. Enzalutamide pretreatment resulted in a decrease in DSB repair ability and re-sensitized 1245C HSD3B1 PCa cells to radiation. Conclusions: 1245C HSD3B1 fuels prostate cancer treatment resistance by elevating regional androgen biosynthesis from adrenal steroid precursors, leading to DDR gene overexpression and enhanced DNA DSB repair kinetics. This work supports clinical validation of biomarker informed selective intensification strategies, e.g., combined androgen blockade, for high-risk men with the 1245C HSD3B1 allele receiving curative intent radiotherapy.

The Maverick trial: A phase 2 study of abivertinib in patients (pts) with metastatic castration resistant prostate cancer (mCRPC).

TPS5106 Background: Abivertinib is a potent, small molecule third-generation tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), Bruton tyrosine kinase (BTK) receptor, and bone marrow tyrosine kinase gene on chromosome X (BMX). Abivertinib irreversibly binds to the BTK receptor, preventing the phosphorylation of the receptor, and also BMX. The adrenal-permissive HSD3B1(1245C) allele, which is present in upwards of 50% of men with prostate cancer, encodes a 3βHSD1 enzyme missense that up-regulates the rate-limiting step of androgen biosynthesis from extragonadal precursor steroids and promotes poor clinical outcomes. 3βHSD1 must be phosphorylated by BMX for androgen biosynthesis. Preclinical studies suggest BMX inhibition blocks 3βHSD1, androgen biosynthesis and CRPC in xenograft models. These provide the rationale for use of the combination of abivertinib and abiraterone to block residual androgen synthesis that escapes abiraterone inhibition. Methods: The Maverick Trial is a multicenter, open-label phase 2 study within the US Dept of Defense Prostate Cancer Clinical Trials Consortium (DOD PCCTC) of abivertinib with abiraterone in pts with mCRPC harboring the adrenal-permissive HSD3B1(1245C) allele (germline heterozygous or homozygous). HSD3B1 status will be confirmed centrally prior to enrollment via CLIA testing at the Cleveland Clinic. The study includes two cohorts: abiraterone-naïve and abiraterone-progressing. There will be cap of 50% of accrual in each cohort for pts heterozygous for the HSD3B1(1245C) allele. An interim futility analysis is embedded in each cohort, and toxicity rates will be continuously monitored for early stoppage according to predefined rules. Pts receive continuous treatment with abivertinib 200 mg BID with abiraterone 1000 mg QD + prednisone 5 mg BID until radiographic progression, unacceptable toxicity, intercurrent illness, or other reason, such as subject withdrawal. The primary endpoint is 6-month rPFS defined as percent of subjects alive and without progression by RECIST version 1.1 for measurable disease and PCWG3 criteria for bone metastases. Secondary and exploratory endpoints include objective response rate, duration of response, PSA progression, pharmacokinetics, pharmacodynamic tissue changes, and mechanisms of response and resistance to therapy via sequential tissue and blood sampling. The study is currently open at 1 site and will activate at 9 additional sites. It is sponsored by Sorrento Therapeutics, Inc, and managed by the Prostate Cancer Clinical Trials Consortium. Trial Registration: NCT05361915 Funding Source: Sorrento Therapeutics, Inc, the trial’s sponsor who is funding the trial. Clinical trial information: NCT05361915 .

PREPARATION CHOLESTEROL AND EFFECTS OF CHOLESTEROL PARADOX: A REVIEW

Cholesterol is the most significant sterol in mammals, which contributes to preserving plasma membrane fluidity and serves as a precursor of oxysterols, bile acids, and steroid hormones. Plasma membranes contain large amounts of cholesterol, which interacts with other molecules in the membrane in various ways. Cholesterol, one of the most significant structural elements, is regarded as a lipid-type molecule. It plays a crucial role in life, primarily in cell membranes and as a precursor to the biosynthesis of numerous steroid hormones in cell membranes. Cholesterol absorption is linked to recurrent cardiovascular events, and patients with higher cholesterol absorption experienced less of a statin's favourable effects. One of the essential and prevalent conditions that affect people's health worldwide is coronary artery disease (CAD), and its incidence has grown over time. The best predictor and moderator of coronary artery disease is the low-density lipoprotein cholesterol plasma level. Higher than lower HDL cholesterol levels were associated with a lower risk of major cardiovascular events. Contrarily in cholesterol paradox states, individuals with advanced HF often have low cholesterol, associated with a poor prognosis and hypercholesterolemia, a key risk factor for CAD. On average, patients with a total cholesterol level of 232 mg/dl had a 25% higher survival rate than those with a total cholesterol level of 193 mg/dl who were suffering from heart failure. A total cholesterol level under 200 mg/dl is generally preferred.

Metabotypes of congenital adrenal hyperplasia in infants determined by gas chromatography-mass spectrometry in spot urine

Biochemical monitoring of treatment in infants with classic congenital adrenal hyperplasia (CAH) is not yet well defined. The aim of this study was to perform a cluster analysis of the urinary steroid metabolome for treatment monitoring of infants with classic salt-wasting CAH. We analyzed spot urine samples obtained from 60 young children ≤ 4 years of age (29 females) with classic CAH due to 21-hydroxylase deficiency treated with hydrocortisone and fludrocortisone by targeted gas chromatography-mass spectrometry (GC-MS). Patients were classified into different groups according to their metabolic patterns (metabotypes) using unsupervised k-means clustering algorithms. Three metabotypes could be discovered. Metabotype #1 (N = 15 (25%)) showed high concentrations of androgen and 17-hydroxyprogesterone (17OHP) precursor steroids, metabotype #2 (N = 28 (47%)) revealed balanced metabolic control, and metabotype #3 (N = 17; 28%) demonstrated severe adrenal suppression with low concentrations of androgen and 17OHP precursor steroids. Daily hydrocortisone doses and urinary concentrations of cortisol and cortisone metabolites did not differ between all three metabotypes. Metabotype #2 had highest daily dose of fludrocortisone (p = 0.006). Receiver operating characteristic curve analysis showed that 11-ketopregnanetriol (area under the curve [AUC] 0.967) and pregnanetriol (AUC 0.936) were most suitable of separating metabotype #1 from #2. For separation between metabotypes #2 vs. #3, the 11-oxygenated androgen metabolite 11-hydroxyandrosterone (AUC 0.983) and the ratio of 11-hydroxyandrosterone to tetrahydrocortisone (AUC 0.970) were most suitable. In conclusion, GC-MS-based urinary steroid metabotyping is a new method to help monitor the treatment of infants with CAH. This method allows classification of under-, over- and adequately treated young children.