Cellular landscape of adrenocortical carcinoma at single-nuclei resolution.

Adrenocortical carcinoma (ACC) is a rare yet devastating tumour of the adrenal gland with a molecular pathology that remains incompletely understood. To gain novel insights into the cellular landscape of ACC, we generated single-nuclei RNA sequencing (snRNA-seq) data sets from twelve ACC tumour samples and analysed these alongside snRNA-seq data sets from normal adrenal glands (NAGs). We find the ACC tumour microenvironment to be relatively devoid of immune cells compared to NAG tissues, consistent with known high tumour purity values for ACC as an immunologically "cold" tumour. Our analysis identifies three separate groups of ACC samples that are characterised by different relative compositions of adrenocortical cell types. These include cell populations that are specifically enriched in the most clinically aggressive and hormonally active tumours, displaying hallmarks of reorganised cell mechanobiology and dysregulated steroidogenesis, respectively. We also identified and validated a population of mitotically active adrenocortical cells that strongly overexpress genes POLQ, DIAPH3 and EZH2 to support tumour expansion alongside an LGR4+ progenitor-like or cell-of-origin candidate for adrenocortical carcinogenesis. Trajectory inference suggests the fate adopted by malignant adrenocortical cells upon differentiation is associated with the copy number or allelic balance state of the imprinted DLK1/MEG3 genomic locus, which we verified by assessing bulk tumour DNA methylation status. In conclusion, our results therefore provide new insights into the clinical and cellular heterogeneity of ACC, revealing how genetic perturbations to healthy adrenocortical renewal and zonation provide a molecular basis for disease pathogenesis.

Predisposing factors for adrenal crisis in chronic adrenal insufficiency: a case-control study.

OBJECTIVE: This study aims to identify susceptibility markers for adrenal crises (AC) in educated patients with chronic adrenal insufficiency (AI). DESIGN: A case-control study involving 66 patients with AI analyzing the impact of glucocorticoid and mineralocorticoid exposure, adrenomedullary function, inflammatory parameters, and educational status on AC frequency. Patients were categorized into low (n = 32) and high (n = 34) AC frequency groups based on AC occurrence (below or 2 times above the average of the reported AC frequency of 8.3 AC/100 patient-years in a previous prospective study). METHODS: Parameters, including cortisol plasma profile and urinary steroid excretion after administration of the morning glucocorticoid dose, 24-h urinary steroid profiling, salivary cortisol profiling, and hair cortisol, estimated cortisol exposure. Polymorphisms (single nucleotide polymorphism [SNP]) of the glucocorticoid receptor (NR3C1) and mineralocorticoid receptor (NR3C2) associated with individual steroid sensitivity were assessed together with SNPs for 11ß-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11ß-hydroxysteroid dehydrogenase 2 (HSD11B2). Mineralocorticoid replacement was evaluated by serum and urinary electrolytes and osmolality, plasma-renin concentration, and ambulatory blood pressure levels. We additionally measured plasma and urinary catecholamines, serum levels of IL6 and hsCRP, and SNPs of IL6 and TNF-alpha. Patient knowledge of AC prevention was assessed by questionnaires. RESULTS: Frequent AC patients had higher daily glucocorticoid doses and hair cortisol levels, with no significant differences in other parameters investigated. AC frequency is inversely correlated with the frequency of self-reported adjustments of the glucocorticoid replacement. CONCLUSION: Higher glucocorticoid dosages in high-risk patients, despite unaffected cortisol metabolism, may be linked to decreased cortisol sensitivity or impaired glucocorticoid absorption. Proactive dose adjustments show a protective effect against AC, regardless of biological vulnerability.

MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

Uncovering associations between mental illness diagnosis, nitric oxide synthase gene variation, and peripheral nitric oxide concentration.

Nitric oxide (NO) signalling has been implicated in the pathogenesis of several mental illnesses; however, its specific contribution remains unclear. We investigated whether peripheral NO concentration is associated with specific diagnoses, and whether there is a correlation with genetic variation in NO synthase (NOS) genes. We included 185 participants in the study; 52 healthy controls, 43 major depressive disorder (MDD) patients, 41 bipolar disorder (BPD) patients, and 49 schizophrenia (SCZ) patients. Clinical, genetic, and biochemical data were collected at admission to a psychiatric hospital and at discharge. Serum was used to quantify concentration of the stable NO metabolites nitrite and nitrate. Individuals were genotyped for the NOS1 exon 1f variable number of tandem repeats 1 (VNTR1) polymorphism, and single nucleotide polymorphisms (SNPs) in the NOS1, NOS1AP and NOS3 genes. At admission, SCZ patients were found to have significantly higher peripheral NO metabolite (NOx-) concentrations compared to healthy controls, MDD and BPD patients. NOS1 exon 1f VNTR1 short allele carriers were found to have significantly increased NOx- concentration. Moreover, this result was still significant in patients even at discharge. The data also revealed that patients who did not remit in their depressive symptoms had significantly increased NOx- concentration compared to remitters at discharge, supported by the finding of a significant positive correlation between depression symptom severity and NOx- concentration. Taken together, it is possible that elevated peripheral NOx- concentration is associated with increased severity of psychopathology, potentially due to NOS1 exon1f VNTR1 genotype. Our results further implicate NO signalling in mental illness pathogenesis, supporting its possible use as a peripheral biomarker, and imply that NOS genotype may play a significant role in regulating peripheral NOx- concentration.

A novel patient-derived cell line of adrenocortical carcinoma shows a pathogenic role of germline MUTYH mutation and high tumour mutational burden.

BACKGROUND: The response of advanced adrenocortical carcinoma (ACC) to current chemotherapies is unsatisfactory and a limited rate of response to immunotherapy was observed in clinical trials. High tumour mutational burden (TMB) and the presence of a specific DNA signature are characteristic features of tumours with mutations in the gene MUTYH encoding the mutY DNA glycosylase. Both have been shown to potentially predict the response to immunotherapy. High TMB in an ACC cell line model has not been reported yet. DESIGN AND METHODS: The JIL-2266 cell line was established from a primary ACC tumour, comprehensively characterised and oxidative damage, caused by a dysfunctional mutY DNA glycosylase, confirmed. RESULTS: Here, we characterise the novel patient-derived ACC cell line JIL-2266, which is deficient in mutY-dependent DNA repair. JIL-2266 cells have a consistent STR marker profile that confirmed congruousness with primary ACC tumour. Cells proliferate with a doubling time of 41 ± 13 h. Immunohistochemistry revealed positivity for steroidogenic factor-1. Mass spectrometry did not demonstrate significant steroid hormone synthesis. JIL-2266 have hemizygous mutations in the tumour suppressor gene TP53 (c.859G>T:p.E287X) and MUTYH (c.316C>T:p.R106W). Exome sequencing showed 683 single nucleotide variants and 4 insertions/deletions. We found increased oxidative DNA damage in the cell line and the corresponding primary tumour caused by impaired mutY DNA glycosylase function and accumulation of 8-oxoguanine. CONCLUSION: This model will be valuable as a pre-clinical ACC cell model with high TMB and a tool to study oxidative DNA damage in the adrenal gland.

PKA Cα subunit mutation triggers caspase-dependent RIIß subunit degradation via Ser114 phosphorylation.

Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing's syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIß protein levels; however, the mechanisms leading to reduced RIIß levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser114 phosphorylation of RIIß is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIß protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing's syndrome.

Coagulation factor XIII activity predicts left ventricular remodelling after acute myocardial infarction.

AIMS: Acute myocardial infarction (MI) is the major cause of chronic heart failure. The activity of blood coagulation factor XIII (FXIIIa) plays an important role in rodents as a healing factor after MI, whereas its role in healing and remodelling processes in humans remains unclear. We prospectively evaluated the relevance of FXIIIa after acute MI as a potential early prognostic marker for adequate healing. METHODS AND RESULTS: This monocentric prospective cohort study investigated cardiac remodelling in patients with ST-elevation MI and followed them up for 1 year. Serum FXIIIa was serially assessed during the first 9 days after MI and after 2, 6, and 12 months. Cardiac magnetic resonance imaging was performed within 4 days after MI (Scan 1), after 7 to 9 days (Scan 2), and after 12 months (Scan 3). The FXIII valine-to-leucine (V34L) single-nucleotide polymorphism rs5985 was genotyped. One hundred forty-six patients were investigated (mean age 58 ± 11 years, 13% women). Median FXIIIa was 118% (quartiles, 102-132%) and dropped to a trough on the second day after MI: 109% (98-109%; P < 0.001). FXIIIa recovered slowly over time, reaching the baseline level after 2 to 6 months and surpassed baseline levels only after 12 months: 124% (110-142%). The development of FXIIIa after MI was independent of the genotype. FXIIIa on Day 2 was strongly and inversely associated with the relative size of MI in Scan 1 (Spearman's ρ = -0.31; P = 0.01) and Scan 3 (ρ = -0.39; P < 0.01) and positively associated with left ventricular ejection fraction: ρ = 0.32 (P < 0.01) and ρ = 0.24 (P = 0.04), respectively. CONCLUSIONS: FXIII activity after MI is highly dynamic, exhibiting a significant decline in the early healing period, with reconstitution 6 months later. Depressed FXIIIa early after MI predicted a greater size of MI and lower left ventricular ejection fraction after 1 year. The clinical relevance of these findings awaits to be tested in a randomized trial.

Neural correlates of NOS1 ex1f-VNTR allelic variation in panic disorder and agoraphobia during fear conditioning and extinction in fMRI.

Neuronal nitric oxide synthase (NOS-I) impacts on fear/anxiety-like behavior in animals. In humans, the short (S) allele of a functional promotor polymorphism of NOS1 (NOS1 ex1f-VNTR) has been shown to be associated with higher anxiety and altered fear conditioning in healthy subjects in the amygdala and hippocampus (AMY/HIPP). Here, we explore the role of NOS1 ex1f-VNTR as a pathophysiological correlate of panic disorder and agoraphobia (PD/AG). In a sub-sample of a multicenter cognitive behavioral therapy (CBT) randomized controlled trial in patients with PD/AG (n = 48: S/S-genotype n=15, S/L-genotype n=21, L/L-genotype n=12) and healthy control subjects, HS (n = 34: S/S-genotype n=7, S/L-genotype n=17, L/L-genotype=10), a differential fear conditioning and extinction fMRI-paradigm was used to investigate how NOS1 ex1f-VNTR genotypes are associated with differential neural activation in AMY/HIPP. Prior to CBT, L/L-allele carriers showed higher activation than S/S-allele carriers in AMY/HIPP. A genotype × diagnosis interaction revealed that the S-allele in HS was associated with a pronounced deactivation in AMY/HIPP, while patients showed contrary effects. The interaction of genotype × stimulus type (CS+, conditioned stimulus associated with an aversive stimulus vs. CS-, unassociated) showed effects on differential learning in AMY/HIPP. All effects were predominately found during extinction. Genotype associated effects in patients were not altered after CBT. Low statistical power due to small sample size in each subgroup is a major limitation. However, our findings provide first preliminary evidence for dysfunctional neural fear conditioning/extinction associated with NOS1 ex1f-VNTR genotype in the context of PD/AG, shedding new light on the complex interaction between genetic risk, current psychopathology and treatment-related effects.

Effects of Germline CYP2W1*6 and CYP2B6*6 Single Nucleotide Polymorphisms on Mitotane Treatment in Adrenocortical Carcinoma: A Multicenter ENSAT Study.

Mitotane is the only approved drug for advanced adrenocortical carcinoma (ACC) and no biomarkers are available to predict attainment of therapeutic plasma concentrations and clinical response. Aim of the study was to evaluate the suitability of cytochrome P450(CYP)2W1 and CYP2B6 single nucleotide polymorphisms (SNPs) as biomarkers. A multicenter cohort study including 182 ACC patients (F/M = 121/61) treated with mitotane monotherapy after radical resection (group A, n = 103) or in not completely resectable, recurrent or advanced disease (group B, n = 79) was performed. CYP2W1*2, CYP2W1*6, CYP2B6*6 and CYP2B6 rs4803419 were genotyped in germline DNA. Mitotane blood levels were measured regularly. Response to therapy was evaluated as time to progression (TTP) and disease control rate (DCR). Among investigated SNPs, CYP2W1*6 and CYP2B6*6 correlated with mitotane treatment only in group B. Patients with CYP2W1*6 (n = 21) achieved less frequently therapeutic mitotane levels (>14 mg/L) than those with wild type (WT) allele (76.2% vs 51.7%, p = 0.051) and experienced shorter TTP (HR = 2.10, p = 0.019) and lower DCR (chi-square = 6.948, p = 0.008). By contrast, 55% of patients with CYP2B6*6 vs. 28.2% WT (p = 0.016) achieved therapeutic range. Combined, a higher rate of patients with CYP2W1*6WT+CYP2B6*6 (60.6%) achieved mitotane therapeutic range (p = 0.034). In not completely resectable, recurrent or advanced ACC, CYP2W1*6 SNP was associated with a reduced probability to reach mitotane therapeutic range and lower response rates, whereas CYP2B6*6 correlated with higher mitotane levels. The association of these SNPs may predict individual response to mitotane.

Prognostic Relevance of Steroid Sulfation in Adrenocortical Carcinoma Revealed by Molecular Phenotyping Using High-Resolution Mass Spectrometry Imaging.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare tumor with variable prognosis even within the same tumor stage. Cancer-related sex hormones and their sulfated metabolites in body fluids can be used as tumor markers. The role of steroid sulfation in ACC has not yet been studied. MALDI mass spectrometry imaging (MALDI-MSI) is a novel tool for tissue-based chemical phenotyping. METHODS: We performed phenotyping of formalin-fixed, paraffin-embedded tissue samples from 72 ACC by MALDI-MSI at a metabolomics level. RESULTS: Tumoral steroid hormone metabolites-estradiol sulfate [hazard ratio (HR) 0.26; 95% CI, 0.10-0.69; P = 0.005] and estrone 3-sulfate (HR 0.22; 95% CI, 0.07-0.63; P = 0.003)-were significantly associated with prognosis in Kaplan-Meier analyses and after multivariable adjustment for age, tumor stage, and sex (HR 0.29; 95% CI, 0.11-0.79; P = 0.015 and HR 0.30; 95% CI, 0.10-0.91; P = 0.033, respectively). Expression of sulfotransferase SULT2A1 was associated with prognosis to a similar extent and was validated to be a prognostic factor in two published data sets. We discovered the presence of estradiol-17ß 3,17-disulfate (E2S2) in a subset of tumors with particularly poor overall survival. Electron microscopy revealed novel membrane-delimited organelles in only these tumors. By applying cluster analyses of metabolomic data, 3 sulfation-related phenotypes exhibited specific metabolic features unrelated to steroid metabolism. CONCLUSIONS: MALDI-MSI provides novel insights into the pathophysiology of ACC. Steroid hormone sulfation may be used for prognostication and treatment stratification. Sulfation-related metabolic reprogramming may be of relevance also in conditions beyond the rare ACC and can be directly investigated by the use of MALDI-MSI.

Driver mutations in USP8 wild-type Cushing's disease.

BACKGROUND: Medical treatment in Cushing's disease (CD) is limited due to poor understanding of its pathogenesis. Pathogenic variants of ubiquitin specific peptidase 8 (USP8) have been confirmed as causative in around half of corticotroph tumors. We aimed to further characterize the molecular landscape of those CD tumors lacking USP8 mutations in a large cohort of patients. METHODS: Exome sequencing was performed on 18 paired tumor-blood samples with wild-type USP8 status. Candidate gene variants were screened by Sanger sequencing in 175 additional samples. The most frequent variant was characterized by further functional in vitro assays. RESULTS: Recurrent somatic hotspot mutations in another deubiquitinase, USP48, were found in 10.3% of analyzed samples. Several possibly damaging variants were found in TP53 in 6 of 18 samples. USP48 variants were associated with smaller tumors and trended toward higher frequency in female patients. They also changed the structural conformation of USP48 and increased its catalytic activity toward its physiological substrates histone 2A and zinc finger protein Gli1, as well as enhanced the stimulatory effect of corticotropin releasing hormone (CRH) on pro-opiomelanocortin production and adrenocorticotropic hormone secretion. CONCLUSIONS: USP48 pathogenic variants are relatively frequent in USP8 wild-type tumors and enhance CRH-induced hormone production in a manner coherent with sonic hedgehog activation. In addition, TP53 pathogenic variants may be more frequent in larger CD tumors than previously reported.

Impact of USP8 Gene Mutations on Protein Deregulation in Cushing Disease.

CONTEXT: Cushing disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8 hyperactive and prevent client proteins from degradation. OBJECTIVE: To investigate the impact of USP8 mutations on proteins deregulated in CD. DESIGN: One hundred eight pituitary adenomas (75 corticotroph [58 USP8 wild type (WT) and 17 USP8 mutated], 14 somatotroph, and 19 nonfunctioning) were investigated by immunohistochemistry. All evaluated proteins [USP8, arginine vasopressin receptor 1b and 2, corticotropin-releasing hormone receptor, cAMP response element-binding protein (CREB), p27/kip1, cyclin E, heat shock protein 90 (HSP90), orphan nuclear receptor 4, epidermal growth factor receptor, histone deacetylase 2, glucocorticoid receptor, cyclin-dependent kinase 5 and Abelson murine leukemia viral oncogene homolog 1 enzyme substrate 1] were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot. RESULTS: Whereas most of the investigated proteins were not differentially expressed, the cell-cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0 ± 1.0 vs 1.1 ± 1.1 in WT adenomas; P = 0.004). In contrast, the chaperone HSP90 was expressed higher (0.5 ± 0.4 vs 0.2 ± 0.4; P = 0.29), and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5 ± 0.40.9 vs 0.70.5 ± 0.40.7; P = 0.014). Accordingly, AtT20 cells transfected with the USP8 P720R mutant had higher phosphorylated CREB (pCREB) levels than WT transfected cells (1.3 ± 0.14 vs 1 ± 0.23; P = 0.13). CONCLUSIONS: We could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and pCREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.

LOX-catalyzed collagen stabilization is a proximal cause for intrinsic resistance to chemotherapy.

The potential of altering the tumor ECM to improve drug response remains fairly unexplored. To identify targets for modification of the ECM aiming to improve drug response and overcome resistance, we analyzed expression data sets from pre-treatment patient cohorts. Cross-evaluation identified a subset of chemoresistant tumors characterized by increased expression of collagens and collagen-stabilizing enzymes. We demonstrate that strong collagen expression and stabilization sets off a vicious circle of self-propagating hypoxia, malignant signaling, and aberrant angiogenesis that can be broken by an appropriate auxiliary intervention: Interfering with collagen stabilization by inhibition of lysyl oxidases significantly enhanced response to chemotherapy in various tumor models, even in metastatic disease. Inhibition of collagen stabilization by itself can reduce or enhance tumor growth depending on the tumor type. The mechanistical basis for this behavior is the dependence of the individual tumor on nutritional supply on one hand and on high tissue stiffness for FAK signaling on the other.

The HDM2 (MDM2) Inhibitor NVP-CGM097 Inhibits Tumor Cell Proliferation and Shows Additive Effects with 5-Fluorouracil on the p53-p21-Rb-E2F1 Cascade in the p53wild type Neuroendocrine Tumor Cell Line GOT1.

BACKGROUND/AIMS: The tumor suppressor p53 is depleted in many tumor cells by the E3 ubiquitin ligase mouse double minute 2 homolog (MDM2) through MDM2/p53 interaction. A novel target for inhibiting p53 degradation and for causing reexpression of p53wild type is inhibition of MDM2. The small molecule NVP-CGM097 is a novel MDM2 inhibitor. We investigated MDM2 inhibition as a target in neuroendocrine tumor cells in vitro. METHODS: Human neuroendocrine tumor cell lines from the pancreas (BON1), lung (NCI-H727), and midgut (GOT1) were incubated with the MDM2 inhibitor NVP-CGM097 (Novartis) at concentrations from 4 to 2,500 nM. RESULTS: While p53wild type GOT1 cells were sensitive to NVP-CGM097, p53mutated BON1 and p53mutated NCI-H727 cells were resistant to NVP-CGM097. Incubation of GOT1 cells with NVP-CGM097 at 100, 500, and 2,500 nM for 96 h caused a significant decline in cell viability to 84.9 ± 9.2% (p < 0.05), 77.4 ± 6.6% (p < 0.01), and 47.7 ± 9.2% (p < 0.01). In a Western blot analysis of GOT1 cells, NVP-CGM097 caused a dose-dependent increase in the expression of p53 and p21 tumor suppressor proteins and a decrease in phospho-Rb and E2F1. Experiments of co-incubation of NVP-CGM097 with 5-fluorouracil, temozolomide, or everolimus each showed additive antiproliferative effects in GOT1 cells. NVP-CGM097 and 5-fluorouracil increased p53 and p21 expression in an additive manner. CONCLUSIONS: MDM2 inhibition seems a promising novel therapeutic target in neuroendocrine tumors harboring p53wild type. Further investigations should examine the potential role of MDM2 inhibitors in neuroendocrine tumor treatment.

Lack of Ubiquitin Specific Protease 8 (USP8) Mutations in Canine Corticotroph Pituitary Adenomas.

PURPOSE: Cushing's disease (CD), also known as pituitary-dependent hyperadrenocorticism, is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. Affected humans and dogs have similar clinical manifestations, however, the incidence of the canine disease is thousand-fold higher. This makes the dog an obvious model for studying the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite certain similarities identified at the molecular level, the question still remains whether the two species have a shared oncogenetic background. Recently, hotspot recurrent mutations in the gene encoding for ubiquitin specific protease 8 (USP8) have been identified as the main driver behind the formation of ACTH-secreting pituitary adenomas in humans. In this study, we aimed to verify whether USP8 mutations also play a role in the development of such tumours in dogs. METHODS: Presence of USP8 mutations was analysed by Sanger and PCR-cloning sequencing in 38 canine ACTH-secreting adenomas. Furthermore, the role of USP8 and EGFR protein expression was assessed by immunohistochemistry in a subset of 25 adenomas. RESULTS: None of the analysed canine ACTH-secreting adenomas presented mutations in the USP8 gene. In a subset of these adenomas, however, we observed an increased nuclear expression of USP8, a phenotype characteristic for the USP8 mutated human tumours, that correlated with smaller tumour size but elevated ACTH production in those tumours. CONCLUSIONS: Canine ACTH-secreting pituitary adenomas lack mutations in the USP8 gene suggesting a different genetic background of pituitary tumourigenesis in dogs. However, elevated nuclear USP8 protein expression in a subset of tumours was associated with a similar phenotype as in their human counterparts, indicating a possible end-point convergence of the different genetic backgrounds in the two species. In order to establish the dog as a useful animal model for the study of CD, further comprehensive studies are needed.

Landscape of somatic mutations in sporadic GH-secreting pituitary adenomas.

CONTEXT: Alterations in the cAMP signaling pathway are common in hormonally active endocrine tumors. Somatic mutations at GNAS are causative in 30-40% of GH-secreting adenomas. Recently, mutations affecting the USP8 and PRKACA gene have been reported in ACTH-secreting pituitary adenomas and cortisol-secreting adrenocortical adenomas respectively. However, the pathogenesis of many GH-secreting adenomas remains unclear. AIM: Comprehensive genetic characterization of sporadic GH-secreting adenomas and identification of new driver mutations. DESIGN: Screening for somatic mutations was performed in 67 GH-secreting adenomas by targeted sequencing for GNAS, PRKACA, and USP8 mutations (n=31) and next-generation exome sequencing (n=36). RESULTS: By targeted sequencing, known activating mutations in GNAS were detected in five cases (16.1%), while no somatic mutations were observed in both PRKACA and USP8. Whole-exome sequencing identified 132 protein-altering somatic mutations in 31/36 tumors with a median of three mutations per sample (range: 1-13). The only recurrent mutations have been observed in GNAS (31.4% of cases). However, seven genes involved in cAMP signaling pathway were affected in 14 of 36 samples and eight samples harbored variants in genes involved in the calcium signaling or metabolism. At the enrichment analysis, several altered genes resulted to be associated with developmental processes. No significant correlation between genetic alterations and the clinical data was observed. CONCLUSION: This study provides a comprehensive analysis of somatic mutations in a large series of GH-secreting adenomas. No novel recurrent genetic alterations have been observed, but the data suggest that beside cAMP pathway, calcium signaling might be involved in the pathogenesis of these tumors.

On the role of NOS1 ex1f-VNTR in ADHD-allelic, subgroup, and meta-analysis.

Attention deficit/ hyperactivity disorder (ADHD) is a heritable neurodevelopmental disorder featuring complex genetics with common and rare variants contributing to disease risk. In a high proportion of cases, ADHD does not remit during adolescence but persists into adulthood. Several studies suggest that NOS1, encoding nitric oxide synthase I, producing the gaseous neurotransmitter NO, is a candidate gene for (adult) ADHD. We here extended our analysis by increasing the original sample, adding two further samples from Norway and Spain, and conducted subgroup and co-morbidity analysis. Our previous finding held true in the extended sample, and also meta-analysis demonstrated an association of NOS1 ex1f-VNTR short alleles with adult ADHD (aADHD). Association was restricted to females, as was the case in the discovery sample. Subgroup analysis on the single allele level suggested that the 21-repeat allele caused the association. Regarding subgroups, we found that NOS1 was associated with the hyperactive/impulsive ADHD subtype, but not to pure inattention. In terms of comorbidity, major depression, anxiety disorders, cluster C personality disorders and migraine were associated with short repeats, in particular the 21-repeat allele. Also, short allele carriers had significantly lower IQ. Finally, we again demonstrated an influence of the repeat on gene expression in human post-mortem brain samples. These data validate the role of NOS-I in hyperactive/impulsive phenotypes and call for further studies into the neurobiological underpinnings of this association. © 2015 Wiley Periodicals, Inc.

Reciprocal regulation of human platelet function by endogenous prostanoids and through multiple prostanoid receptors.

Platelets are permanently exposed to a variety of prostanoids formed by blood cells or the vessel wall. The two major prostanoids, prostacyclin and thromboxane act through well established pathways mediated by their respective G-protein coupled receptors inhibiting or promoting platelet aggregation accordingly. Yet the role of other prostanoids and prostanoid receptors for platelet function regulation has not been thoroughly investigated. We aimed at a comprehensive analysis of prostanoid effects on platelets, the receptors and pathways involved and functional consequences. We analyzed cAMP formation and phosphorylation of proteins pivotal to platelet function as well as functional platelet responses such as secretion, aggregation and phosphorylation. The types of prostanoid receptors contributing and their individual share in signaling pathways were analyzed and indicated a major role for prostanoid IP1 and DP1 receptors followed by prostanoid EP4 and EP3 receptors while prostanoid EP2 receptors appear less relevant. We could show for the first time the reciprocal action of the endogenous prostaglandin PGE2 on platelets by functional responses and phosphorylation events. PGE2 evokes stimulatory as well as inhibitory effects in a concentration dependent manner in platelets via prostanoid EP3 or EP4 and prostanoid DP1 receptors. A mathematical model integrating the pathway components was established which successfully reproduces the observed platelet responses. Additionally we could show that human platelets themselves produce sufficient PGE2 to act in an autocrine or paracrine fashion. These mechanisms may provide a fine tuning of platelet responses in the circulating blood by either promoting or limiting endogenous platelet activation.

Association of a functional variant of the nitric oxide synthase 1 gene with personality, anxiety, and depressiveness.

A functional promoter polymorphism of the nitric oxide synthase 1 gene first exon 1f variable number tandem repeat (NOS1 ex1f-VNTR) is associated with impulsivity and related psychopathology. Facets of impulsivity are strongly associated with personality traits; maladaptive impulsivity with neuroticism; and adaptive impulsivity with extraversion. Both high neuroticism and low extraversion predict anxiety and depressive symptoms. The aim of the present study was to evaluate the effect of the NOS1 ex1f-VNTR genotype and possible interaction with environmental factors on personality, anxiety, and depressiveness in a population-representative sample. Short allele carriers had higher neuroticism and anxiety than individuals with the long/long (l/l) genotype. Male short/short homozygotes also had higher extraversion. In the face of environmental adversity, females with a short allele had higher scores of neuroticism, anxiety, and depressiveness compared to the l/l genotype. Males were more sensitive to environmental conditions when they had the l/l genotype and low extraversion. In conclusion, the NOS1 ex1f-VNTR influences personality and emotional regulation dependent on gender and environment. Together with previous findings on the effect of the NOS1 genotype on impulse control, these data suggest that NOS1 should be considered another plasticity gene, because its variants are associated with different coping strategies.

Frog urinary bladder epithelial cells express TLR4 and respond to bacterial LPS by increase of iNOS expression and L-arginine uptake.

As in mammals, epithelium of the amphibian urinary bladder forms a barrier to pathogen entry and is a first line of defense against penetrating microorganisms. We investigated the effect of Escherichia coli LPS on generation of nitric oxide (NO), a critically important mediator during infectious processes, by primary cultured frog (Rana temporaria) urinary bladder epithelial cells (FUBEC). It was found that FUBEC constitutively express Toll-like receptor 4 (TLR4), a receptor of LPS, and respond to LPS (10 µg/ml) by stimulation of inducible nitric oxide synthase (iNOS) mRNA/protein expression and NOS activity measured by nitrite produced in the culture medium and by citrulline assay. We characterized uptake of l-arginine, a precursor in NO synthesis, by FUBEC and showed that it is mediated mainly by the y+ cationic amino acid transport system. LPS stimulated l-arginine uptake, and this effect was blocked by the iNOS inhibitor 1400W. Arginase II was found to be expressed in FUBEC. Inhibition of arginase activity by (S)-(boronoethyl)-l-cysteine increased generation of NO, suggesting contribution of arginase to NO production via competing with NOS for the substrate. LPS altered neither total arginase activity nor arginase II expression. Among epithelial cells, phagocytic macrophage-like cells were observed, but they did not contribute to LPS-induced NO production. These data demonstrate that amphibian urinary bladder epithelial cells recognize LPS and respond to it by increased generation of NO via stimulation of iNOS expression and l-arginine uptake, which appears to be essential for the regulation of the innate immune response and the inflammation in bladder epithelium.