The long non-coding RNA OTX2-AS1 promotes tumor growth and predicts response to BCL-2 inhibition in medulloblastoma.

PURPOSE: Primary brain tumors are a leading cause of cancer-related death in children, and medulloblastoma is the most common malignant pediatric brain tumor. The current molecular characterization of medulloblastoma is mainly based on protein-coding genes, while little is known about the involvement of long non-coding RNAs (lncRNAs). This study aimed to elucidate the role of the lncRNA OTX2-AS1 in medulloblastoma. METHODS: Analyses of DNA copy number alterations, methylation profiles, and gene expression data were used to characterize molecular alterations of OTX2-AS1 in medulloblastoma tissue samples. In vitro analyses of medulloblastoma cell models and orthotopic in vivo experiments were carried out for functional characterization of OTX2-AS1. High-throughput drug screening was employed to identify pharmacological inhibitors, while proteomics and metabolomics analyses were performed to address potential mechanisms of drug action. RESULTS: We detected amplification and consecutive overexpression of OTX2 and OTX2-AS1 in a subset of medulloblastomas. In addition, OTX2-AS1 promoter methylation was linked to OTX2-AS1 expression. OTX2-AS1 knockout reduced medulloblastoma cell viability and cell migration in vitro and prolonged survival in the D283 orthotopic medulloblastoma mouse xenograft model. Pharmacological inhibition of BCL-2 suppressed the growth of OTX2-AS1 overexpressing medulloblastoma cells in vitro. CONCLUSIONS: Our study revealed a pro-tumorigenic role of OTX2-AS1 in medulloblastoma and identified BCL-2 inhibition as a potential therapeutic approach to target OTX2-AS1 overexpressing medulloblastoma cells.

Metabolomics, machine learning and immunohistochemistry to predict succinate dehydrogenase mutational status in phaeochromocytomas and paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background in over one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and several other tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlighting the importance of identifying SDHx mutations for patient management. Genetic variants of unknown significance, where implications for the patient and family members are unclear, are a problem for interpretation. For such cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB (SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatography-mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions provide an alternative method. Here, we compare SDHB-IHC with metabolite profiling in 189 tumours from 187 PPGL patients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to establish predictive models for interpreting metabolite data. Metabolite profiling showed higher diagnostic specificity compared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite profiles improved predictive ability over that of the SFR, in particular for hard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, the combination of metabolite profiling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classified all but one of the false negatives from metabolite profiling strategies, while metabolite profiling correctly classified all but one of the false negatives/positives from SDHB-IHC. From 186 tumours with confirmed status of SDHx variant pathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benefits of both strategies for patient management. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Intratumoral heterogeneity of MYC drives medulloblastoma metastasis and angiogenesis.

BACKGROUND: Intratumoral heterogeneity is crucially involved in metastasis, resistance to therapy, and cancer relapse. Amplifications of the proto-oncogene MYC display notable heterogeneity at the single-cell level and are associated with a particularly dismal prognosis in high-risk medulloblastomas (MBs). The aim of this study was to establish the relevance of interclonal cross-talk between MYC-driven and non-MYC-driven MB cells. METHODS: We used fluorescence in situ hybridization, single-cell transcriptomics, and immunohistochemistry, in vitro isogenic cell models, non-targeted proteomics, mass spectrometry-based metabolite quantification, HUVECs tube formation assay, and orthotopic in vivo experiments to investigate interclonal cross-talk in MB. RESULTS: We found that the release of lactate dehydrogenase A (LDHA) from MYC-driven cells facilitates metastatic seeding and outgrowth, while secretion of dickkopf WNT signaling pathway inhibitor 3 from non-MYC-driven cells promotes tumor angiogenesis. This tumor-supporting interaction between both subclones was abrogated by targeting the secretome through pharmacological and genetic inhibition of LDHA, which significantly suppressed tumor cell migration. CONCLUSION: Our study reveals the functional relevance of clonal diversity and highlights the therapeutic potential of targeting the secretome to interrupt interclonal communication and progression in high-risk MB.

HIF2α regulates the synthesis and release of epinephrine in the adrenal medulla.

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signaling on adrenal function remains poorly understood. Here, we reveal that deficiency of HIF (hypoxia inducible factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Simultaneous loss of PHD2 in renal erythropoietin (EPO)-producing cells (REPCs) stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis), and systemic hypoglycemia, which is necessary and sufficient to enhance exocytosis of epinephrine from the adrenal medulla. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla regulates the synthesis of epinephrine, whereas in REPCs, it indirectly induces the release of this hormone. Our findings are also highly relevant to the testing of small molecule PHD inhibitors in phase III clinical trials for patients with renal anemia. KEY MESSAGES: HIF2α and not HIF1α modulates PNMT during epinephrine synthesis in chromaffin cells. The PHD2-HIF2α-EPO axis induces erythrocytosis and hypoglycemia. Reduced systemic glucose facilitates exocytosis of epinephrine from adrenal gland.

Targeted Quantification of Carbon Metabolites Identifies Metabolic Progression Markers and an Undiagnosed Case of SDH-Deficient Clear Cell Renal Cell Carcinoma in a German Cohort.

Renal cell carcinoma (RCC) is among the 10 most common cancer entities and can be categorised into distinct subtypes by differential expression of Krebs cycle genes. We investigated the predictive value of several targeted metabolites with regards to tumour stages and patient survival in an unselected cohort of 420 RCCs. Unsupervised hierarchical clustering of metabolite ratios identified two main clusters separated by α-ketoglutarate (α-KG) levels and sub-clusters with differential levels of the oncometabolite 2-hydroxyglutarate (2HG). Sub-clusters characterised by high 2HG were enriched in higher tumour stages, suggesting metabolite profiles might be suitable predictors of tumour stage or survival. Bootstrap forest models based on single metabolite signatures showed that lactate, 2HG, citrate, aspartate, asparagine, and glutamine better predicted the cancer-specific survival (CSS) of clear cell RCC patients, whereas succinate and α-ketoglutarate were better CSS predictors for papillary RCC patients. Additionally, this assay identifies rare cases of tumours with SDHx mutations, which are caused predominantly by germline mutations and which predispose to development of different neoplasms. Hence, analysis of selected metabolites should be further evaluated for potential utility in liquid biopsies, which can be obtained using less invasive methods and potentially facilitate disease monitoring for both patients and caregivers.

Overnight/first-morning urine free metanephrines and methoxytyramine for diagnosis of pheochromocytoma and paraganglioma: is this an option?

OBJECTIVE: Sympathoadrenal activity is decreased during overnight rest. This study assessed whether urinary-free normetanephrine, metanephrine and methoxytyramine in overnight/first-morning urine collections might offer an alternative to measurements in 24-h collections or plasma for diagnosis of pheochromocytoma and paraganglioma (PPGL). DESIGN AND METHODS: Prospective multicenter cross-sectional diagnostic study involving 706 patients tested for PPGL, in whom tumors were confirmed in 79 and excluded in 627 after follow-up. Another 335 age- and sex-matched volunteers were included for reference purposes. Catecholamines and their free O-methylated metabolites were measured in 24-h collections divided according to waking and sleeping hours and normalized to creatinine. Plasma metabolites from blood sampled after supine rest were measured for comparison. RESULTS: Urinary outputs of norepinephrine, normetanephrine, epinephrine and metanephrine in the reference population were respectively 50 (48-52)%, 35 (32-37)%, 76 (74-78)% and 15 (12-17)% lower following overnight than daytime collections. Patients in whom PPGLs were excluded showed 28 (26-30)% and 6 (3-9)% day-to-night falls in normetanephrine and metanephrine, while patients with PPGLs showed no significant day-to-night falls in metabolites. Urinary methoxytyramine was consistently unchanged from day to night. According to receiver-operating characteristic curves, diagnostic accuracy of metabolite measurements in overnight/first-morning urine samples did not differ from measurements in 24-h urine collections, but was lower for both than for plasma. Using optimized reference intervals, diagnostic specificity was higher for overnight than daytime collections at similar sensitivities. CONCLUSIONS: Measurements of urinary-free catecholamine metabolites in first-morning/overnight urine collections offer an alternative for diagnosis of PPGL to 24-h collections but remain less accurate than plasma measurements.

Glucocorticoid Excess in Patients with Pheochromocytoma Compared with Paraganglioma and Other Forms of Hypertension.

CONTEXT: 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. OBJECTIVE: This study addresses the hypothesis that patients with pheochromocytoma and paraganglioma (PPGL) have altered steroid production compared with patients with primary hypertension. DESIGN: Multicenter cross-sectional study. SETTING: Twelve European referral centers. PATIENTS: Subjects included 182 patients with pheochromocytoma, 36 with paraganglioma and 270 patients with primary hypertension. Patients with primary aldosteronism (n = 461) and Cushing syndrome (n = 124) were included for additional comparisons. INTERVENTION: In patients with PPGLs, surgical resection of tumors. OUTCOME MEASURES: Differences in mass spectrometry-based profiles of 15 adrenal steroids between groups and after surgical resection of PPGLs. Relationships of steroids to plasma and urinary metanephrines and urinary catecholamines. RESULTS: Patients with pheochromocytoma had higher (P < .05) circulating concentrations of cortisol, 11-deoxycortisol, 11-deoxycorticosterone, and corticosterone than patients with primary hypertension. Concentrations of cortisol, 11-deoxycortisol, and corticosterone were also higher (P < .05) in patients with pheochromocytoma than with paraganglioma. These steroids correlated positively with plasma and urinary metanephrines and catecholamines in patients with pheochromocytoma, but not paraganglioma. After adrenalectomy, there were significant decreases in cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, aldosterone, and 18-oxocortisol. CONCLUSIONS: This is the first large study in patients with PPGLs that supports in a clinical setting the concept of adrenal cortical-medullary interactions involving an influence of catecholamines on adrenal steroids. These findings could have implications for the cardiovascular complications of PPGLs and the clinical management of patients with the tumors.

Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s-1. This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

Catalytic and hydrodynamic properties of styrene monooxygenases from Rhodococcus opacus 1CP are modulated by cofactor binding.

Styrene monooxygenases (SMOs) are flavoenzymes catalyzing the epoxidation of styrene into styrene oxide. SMOs are composed of a monooxygenase (StyA) and a reductase (StyB). The latter delivers reduced FAD to StyA on the expense of NADH. We identified Rhodococcus opacus 1CP as the first microorganism to possess three different StyA isoforms occurring in two systems StyA1/StyA2B and StyA/StyB, respectively. The hydrodynamic properties of StyA isozymes were found to be modulated by the binding of the (reduced) FAD cofactor. StyA1 and SyA2B mainly occur as dimers in their active forms while StyA is a monomer. StyA1 showed the highest epoxidation activity and excellent enantioselectivity in aromatic sulfoxidation. The hydrodynamic and biocatalytic properties of SMOs from strain 1CP are of relevance for investigation of possible industrial applications.