Somatic mutations have been identified in more than half of aldosterone-producing adenomas (APAs) through mutation hotspot sequencing. The underlying pathogenesis of inappropriate aldosterone synthesis in the remaining population is still unknown.

In recent years it has been recognized that clinical translation of novel therapeutic strategies for patients with adrenocortical carcinoma (ACC) often fails. These disappointing results indicate that the currently utilized tumor models only poorly reflect relevant pathophysiology and, thereby, do not predict clinical applicability of novel pharmacological approaches. However, also the development of new preclinical ACC models has remained a challenge with only one human cell line (NCI-H295R) and one recently established human pediatric xenograft model (SJ-ACC3) being available for this highly heterogeneous malignancy. Our current study furthermore reveals a poor reproducibility of therapeutic action between different clones of the most commonly used tumor model NCI-H295R. In an attempt to broaden the current preclinical armamentarium, we aimed at the development of patient-individual tumor models. During these studies, one xenograft (MUC-1) displayed marked engraftment and sustained tumor growth. MUC-1 tumor analysis revealed highly vascularized, proliferating and SF-1 positive xenografts. In a next step, we characterized all currently available human tumor models for ACC for Ki67, SF-1 and EGF-receptor status in comparison with MUC-1-xenografts. In addition, we established a primary culture, which is now viable over 31 passages with sustained nuclear SF-1 and cytoplasmic 3βHSD immuno-positivity. Subsequent investigation of therapeutic responsiveness upon treatment with the current systemic gold standard EDP-M (etoposide, doxorubicin, cisplatin and mitotane) demonstrated maintenance of the clinically observed drug resistance for MUC-1 exclusively. In summary, we provide evidence for a novel patient-derived tumor model with the potential to improve clinical prediction of novel therapeutic strategies for patients with ACC.

Aldosterone synthase is the key rate-limiting enzyme in adrenal aldosterone production, and induction of its gene (CYP11B2) results in the progression of hypertension. As hypertension is a frequent complication among patients with diabetes, we set out to elucidate the link between diabetes mellitus and hypertension. We examined the effects of high glucose on CYP11B2 expression and aldosterone production using human adrenal H295R cells and a stable H295R cell line expressing a CYP11B2 5'-flanking region/luciferase cDNA chimeric construct. d-glucose (d-glu), but not its enantiomer l-glucose, dose dependently induced CYP11B2 transcription and mRNA expression. A high concentration (450 mg·dL-1) of d-glu time dependently induced CYP11B2 transcription and mRNA expression. Moreover, high glucose stimulated secretion of aldosterone into the media. Transient transfection studies using deletion mutants/nerve growth factor-induced clone B (NGFIB) response element 1 (NBRE-1) point mutant of CYP11B2 5'-flanking region revealed that the NBRE-1 element, known to be activated by transcription factors NGFIB and NURR1, was responsible for the high glucose-mediated effect. High glucose also induced the mRNA expression of these transcription factors, especially that of NURR1, but NURR1 knockdown using its siRNA did not affect high glucose-induced CYP11B2 mRNA expression. Taken together, it is speculated that high glucose may induce CYP11B2 transcription via the NBRE-1 element in its 5'-flanking region, resulting in the increase in aldosterone production although high glucose-induced NURR1 is not directly involved in the effect. Additionally, glucose metabolism and calcium channels were found to be involved in the high glucose effect. Our observations suggest one possible explanation for the high incidence of hypertension in patients with diabetes.

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterone's high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10(-41) and rs6258, p = 2.3×10(-22)). Subjects with ≥ 3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10(-16)). The rs6258 polymorphism in exon 4 of SHBG affected SHBG's affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.

Genetic alterations in signaling pathways that control cell-cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations, copy-number changes, mRNA expression, gene fusions and DNA methylation in 9,125 tumors profiled by The Cancer Genome Atlas (TCGA), we analyzed the mechanisms and patterns of somatic alterations in ten canonical pathways: cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGFβ signaling, p53 and β-catenin/Wnt. We charted the detailed landscape of pathway alterations in 33 cancer types, stratified into 64 subtypes, and identified patterns of co-occurrence and mutual exclusivity. Eighty-nine percent of tumors had at least one driver alteration in these pathways, and 57% percent of tumors had at least one alteration potentially targetable by currently available drugs. Thirty percent of tumors had multiple targetable alterations, indicating opportunities for combination therapy.

In the adrenal gland, neuroendocrine cells that synthesize catecholamines and epithelial cells that produce steroid hormones are united beneath a common organ capsule to function as a single stress-responsive organ. The functional anatomy of the steroid hormone-producing adrenal cortex and the catecholamine-producing medulla is ill defined at the level of small molecules. Here, we report a comprehensive high-resolution mass spectrometry imaging (MSI) map of the normal human adrenal gland. A large variety of biomolecules was accessible by matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI, including nucleoside phosphates indicative of oxidative phosphorylation, sterol and steroid metabolites, intermediates of glycolysis and the tricarboxylic acid cycle, lipids, and fatty acids. Statistical clustering analyses yielded a molecularly defined adrenal anatomy of 10 distinct molecular zones including a highly structured corticomedullary interface. By incorporating pathway information, activities of carbohydrate, amino acid, and lipid metabolism as well as endocrine bioactivity were revealed to be highly spatially organized, which could be visualized as different molecularly defined zones. Together, these findings provide a molecular definition of human adult adrenal gland structure beyond classical histological anatomy.

Catecholamines and adrenocortical steroids are important regulators of blood pressure. Bidirectional relationships between adrenal steroids and catecholamines have been established but whether this is relevant to patients with pheochromocytoma is unclear.

High-throughput sequencing of genomes, exomes, and disease-focused gene panels is becoming increasingly common for molecular diagnostics. However, identifying a single clinically relevant pathogenic variant among thousands of genetic polymorphisms is a challenging task. Publicly available genomic databases are useful resources to filter out common genetic variants present in the population and enable the identification of each disease-causing variant. Based on our experience applying these technologies at Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, Brazil, we recognized that the Brazilian population is not adequately represented in widely available genomic databases.

Adrenocortical cancer is a rare malignant neoplasm associated with a dismal prognosis. Identification of the molecular pathways involved in adrenal tumorigenesis is essential for a better understanding of the disease mechanism and improvement of its treatment. The aim of this study is to define the prevalence of alterations in DNA mismatch repair (MMR) genes in Lynch syndrome among pediatric patients with adrenocortical neoplasia from southern Brazil, where the prevalence of a specific TP53 germline mutation (p.Arg337His) is quite high. Thirty-six pediatric patients were retrospectively evaluated. Immunohistochemistry (IHC) for the MMR enzymes MLH1, MSH2, MSH6, and PMS2, as well as next-generation sequencing (NGS) were performed. For IHC, 36 pediatric tumors were tested. In all of them, the expression of all evaluated MMR proteins was well-preserved. For NGS, 35 patients with pediatric tumor were tested. Three patients (8.57%) with the TP53 p.Arg337His germline mutation presented pathogenic and likely pathogenic variants in the MMR genes (two in MLH1 and one in MSH6). The prevalence of altered MMR genes among pediatric patients was elevated (8.57%) and higher than in colorectal and endometrial cancer cohorts. Pediatric patients with adrenocortical tumors should, thus, be strongly considered as at genetic risk for Lynch syndrome.

Results of previous studies demonstrated clear racial differences in the prevalence of somatic mutations among patients with aldosterone-producing adenoma (APA). For instance, those in East Asian countries have a high prevalence of somatic mutations in KCNJ5, whereas somatic mutations in other aldosterone-driving genes are rare.

Cushing's disease is a rare but devastating and difficult to manage condition. The somatostatin analogue pasireotide is the only pituitary-targeting pharmaceutical approved for the treatment of Cushing's disease but is accompanied by varying efficacy and potentially severe side effects. Finding means to predict which patients are more likely to benefit from this treatment may improve their management. More than half of corticotroph tumours harbour mutations in the USP8 gene, and there is evidence of higher somatostatin receptor 5 (SSTR5) expression in the USP8-mutant tumours. Pasireotide has a high affinity for SSTR5, indicating that these tumours may be more sensitive to treatment. To test this hypothesis, we examined the inhibitory action of pasireotide on adrenocorticotrophic hormone synthesis in primary cultures of human corticotroph tumour with assessed USP8 mutational status and in immortalized murine corticotroph tumour cells overexpressing human USP8 mutants frequent in Cushing's disease. Our in vitro results demonstrate that pasireotide exerts a higher antisecretory response in USP8-mutant corticotroph tumours. Overexpressing USP8 mutants in a murine corticotroph tumour cell model increased endogenous somatostatin receptor 5 (Sstr5) transcription. The murine Sstr5 promoter has two binding sites for the activating protein 1 (AP-1) and USP8 mutants possibly to mediate their action by stimulating AP-1 transcriptional activity. Our data corroborate the USP8 mutational status as a potential marker of pasireotide response and describe a potential mechanism through which USP8 mutants may regulate SSTR5 gene expression.

Due to its rare incidence, molecular features of primary aldosteronism (PA) in young adults are largely unknown. Recently developed targeted mutational analysis identified aldosterone-driver somatic mutations in aldosterone-producing lesions, including aldosterone-producing adenomas (APAs), aldosterone-producing nodules (APNs), and aldosterone-producing micronodules, formerly known as aldosterone-producing cell clusters.

Hypertension is a major global health problem with high prevalence and complex associated health risks. Primary hypertension (PHT) is most common and the reasons behind primary hypertension are largely unknown. Endocrine hypertension (EHT) is another complex form of hypertension with an estimated prevalence varying from 3 to 20% depending on the population studied. It occurs due to underlying conditions associated with hormonal excess mainly related to adrenal tumours and sub-categorised: primary aldosteronism (PA), Cushing's syndrome (CS), pheochromocytoma or functional paraganglioma (PPGL). Endocrine hypertension is often misdiagnosed as primary hypertension, causing delays in treatment for the underlying condition, reduced quality of life, and costly antihypertensive treatment that is often ineffective. This study systematically used targeted metabolomics and high-throughput machine learning methods to predict the key biomarkers in classifying and distinguishing the various subtypes of endocrine and primary hypertension. The trained models successfully classified CS from PHT and EHT from PHT with 92% specificity on the test set. The most prominent targeted metabolites and metabolite ratios for hypertension identification for different disease comparisons were C18:1, C18:2, and Orn/Arg. Sex was identified as an important feature in CS vs. PHT classification.

The importance of pre-existing immune responses to seasonal endemic coronaviruses (HCoVs) for the susceptibility to SARS-CoV-2 infection and the course of COVID-19 is the subject of an ongoing scientific debate. Recent studies postulate that immune responses to previous HCoV infections can either have a slightly protective or no effect on SARS-CoV-2 pathogenesis and, consequently, be neglected for COVID-19 risk stratification. Challenging this notion, we provide evidence that pre-existing, anti-nucleocapsid antibodies against endemic α-coronaviruses and S2 domain-specific anti-spike antibodies against β-coronavirus HCoV-OC43 are elevated in patients with COVID-19 compared to pre-pandemic donors. This finding is particularly pronounced in males and in critically ill patients. Longitudinal evaluation reveals that antibody cross-reactivity or polyclonal stimulation by SARS-CoV-2 infection are unlikely to be confounders. Thus, specific pre-existing immunity to seasonal coronaviruses may increase susceptibility to SARS-CoV-2 and predispose individuals to an adverse COVID-19 outcome, guiding risk management and supporting the development of universal coronavirus vaccines.

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.

Glucocorticoids (GCs) are widely applied anti-inflammatory drugs that are associated with adverse metabolic effects including insulin resistance and weight gain. Previous research indicates that GCs may negatively impact brown adipose tissue (BAT) activity in rodents and humans.

Pheochromocytomas/paragangliomas (PPGLs) with pathogenic mutations in the succinate dehydrogenase subunit B (SDHB) are associated with a high metastatic risk. Somatostatin receptor 2 (SSTR2)-dependent imaging is the most sensitive imaging modality for SDHB-related PPGLs, suggesting that SSTR2 expression is a significant cell surface therapeutic biomarker of such tumors.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1(null) (Prop1(-/-)) and the Ames dwarf (Prop1(df/df)) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism.

Aldosterone is the mineralocorticoid responsible for sodium retention, thus increased blood volume and pressure. Excessive production of aldosterone results in high blood pressure as well as renal disease, stroke, and visual loss via both direct effects and effects on blood pressure. Weight gain is often associated with increased blood pressure, but it remains unclear how obesity increases blood pressure. Obese patients typically have higher lipoprotein levels; moreover, some studies have suggested that aldosterone levels are also elevated and represent a link between obesity and hypertension. Very-low-density lipoprotein (VLDL) functions to transport triglycerides from the liver to peripheral tissues. Although previous studies have demonstrated that VLDL can stimulate aldosterone production, the mechanisms underlying this effect are largely unclear. Here we show for the first time that phospholipase D (PLD) is involved in VLDL-induced aldosterone production in both a human adrenocortical cell line (HAC15) and primary cultures of bovine zona glomerulosa cells. Our data also reveal that PLD mediates steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression via increasing the phosphorylation (activation) of their regulatory transcription factors. Finally, by using selective PLD inhibitors, our studies suggest that both PLD1 and PLD2 isoforms play an important role in VLDL-induced aldosterone production.