GLP-1 and PYY for the treatment of obesity: a pilot study on the use of agonists and antagonists in diet-induced rats.

Objective: Combination therapies with gut hormone analogs represent promising treatment strategies for obesity. This pilot study investigates the therapeutic potential of modulators of the glucagon-like peptide 1 (GLP-1) and neuropeptide Y (NPY) system using GLP-1 receptor agonists (semaglutide) and antagonists (exendin 9-39), as well as non-selective and NPY-Y2-receptor selective peptide tyrosine tyrosine (PYY) analogs (PYY3-36/NNC0165-0020 and NNC0165-1273) and an NPY-Y2 receptor antagonist (JNJ31020028). Methods: High-fat diet (HFD)-induced obese rats were randomized into following treatment groups: group 1, nonselective PYY analog + semaglutide (n = 4); group 2, non-selective and NPY-Y2 receptor selective PYY analog + semaglutide (n = 2); group 3, GLP-1 receptor antagonist + NPY-Y2 receptor antagonist (n = 3); group 4, semaglutide (n = 5); and group 5, control (n = 5). Animals had free access to HFD and low-fat diet. Food intake, HFD preference and body weight were measured daily. Results: A combinatory treatment with a non-selective PYY analog and semaglutide led to a maximum body weight loss of 14.0 ± 4.9% vs 9.9 ± 1.5% with semaglutide alone. Group 2 showed a maximum weight loss of 20.5 ± 2.4%. While HFD preference was decreased in group 2, a strong increase in HFD preference was detected in group 3. Conclusions: PYY analogs (especially NPY-Y2 selective receptor agonists) could represent a promising therapeutic approach for obesity in combination with GLP-1 receptor agonists. Additionally, combined GLP-1 and PYY3-36 receptor agonists might have beneficial effects on food preference.

Association of preclinical blood glucose with hospitalization rate and in-hospital mortality: A single-center retrospective cohort study.

Objective: Critical illness is often accompanied by elevated blood glucose, which generally correlates with increased morbidity and mortality. Prehospital blood glucose (PBG) level might be a useful and easy-to-perform tool for risk assessment in emergency medicine. This retrospective single-center cohort study was designed to analyze the association of prehospital glucose measurements with hospitalization rate and in-hospital mortality. Methods: Records of 970 patients admitted to a university hospital by an emergency physician were analyzed. Patients with a PBG ≥140 mg/dL (G1, n = 394, equal to 7.8 mmol/L) were compared with patients with a PBG <140 mg/dL (G2, n = 576). Multivariable logistic regression models were used to correct for age, prediagnosed diabetes, and sex. Results: Five hundred thirty-four patients (55%) were hospitalized. In comparison to normoglycemic patients, hyperglycemic patients were more likely to be hospitalized with an adjusted odds ratio (OR) of 1.48 (95% confidence interval [CI] 1.11-1.97), more likely to be admitted to the intensive care unit (ICU) with an adjusted OR of 1.74 (95% CI 1.31-2.31) and more likely to die in the hospital with an adjusted OR of 1.84 (95% CI 0.96-3.53). Hospitalized hyperglycemic patients had a median length of stay of 6.0 days (interquartile range [IQR] 8.0) compared to 3.0 days (IQR 6.0) in the normoglycemic group (P < 0.001). In the subgroup analysis of cases without known diabetes, patients with PBG ≥140 mg/dL were more likely to be hospitalized with an adjusted OR of 1.49 (95% CI 1.10-2.03) and more likely to be admitted to ICU/intermediate care with an adjusted OR of 1.80 (95% CI 1.32-2.45), compared to normoglycemic patients. Conclusion: Elevated PBG ≥140 mg/dL was associated with a higher hospitalization risk, a longer length of stay, and a higher mortality risk and may therefore be included in risk assessment scores.

Improving susceptibility of neuroendocrine tumors to radionuclide therapies: personalized approaches towards complementary treatments.

Radionuclide therapies are an important tool for the management of patients with neuroendocrine neoplasms (NENs). Especially [131I]MIBG and [177Lu]Lu-DOTA-TATE are routinely used for the treatment of a subset of NENs, including pheochromocytomas, paragangliomas and gastroenteropancreatic tumors. Some patients suffering from other forms of NENs, such as medullary thyroid carcinoma or neuroblastoma, were shown to respond to radionuclide therapy; however, no general recommendations exist. Although [131I]MIBG and [177Lu]Lu-DOTA-TATE can delay disease progression and improve quality of life, complete remissions are achieved rarely. Hence, better individually tailored combination regimes are required. This review summarizes currently applied radionuclide therapies in the context of NENs and informs about recent advances in the development of theranostic agents that might enable targeting subgroups of NENs that previously did not respond to [131I]MIBG or [177Lu]Lu-DOTA-TATE. Moreover, molecular pathways involved in NEN tumorigenesis and progression that mediate features of radioresistance and are particularly related to the stemness of cancer cells are discussed. Pharmacological inhibition of such pathways might result in radiosensitization or general complementary antitumor effects in patients with certain genetic, transcriptomic, or metabolic characteristics. Finally, we provide an overview of approved targeted agents that might be beneficial in combination with radionuclide therapies in the context of a personalized molecular profiling approach.

Obesity and its comorbidities, current treatment options and future perspectives: Challenging bariatric surgery?

Obesity and its comorbidities, including type 2 diabetes mellitus, cardiovascular disease, heart failure and non-alcoholic liver disease are a major health and economic burden with steadily increasing numbers worldwide. The need for effective pharmacological treatment options is strong, but, until recently, only few drugs have proven sufficient efficacy and safety. This article provides a comprehensive overview of obesity and its comorbidities, with a special focus on organ-specific pathomechanisms. Bariatric surgery as the so far most-effective therapeutic strategy, current pharmacological treatment options and future treatment strategies will be discussed. An increasing knowledge about the gut-brain axis and especially the identification and physiology of incretins unfolds a high number of potential drug candidates with impressive weight-reducing potential. Future multi-modal therapeutic concepts in obesity treatment may surpass the effectivity of bariatric surgery not only with regard to weight loss, but also to associated comorbidities.

Metastatic Pheochromocytoma and Paraganglioma: Somatostatin Receptor 2 Expression, Genetics, and Therapeutic Responses.

CONTEXT: Pheochromocytomas and 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. OBJECTIVE: Exploration of the relationship between SSTR2 immunoreactivity and SDHB immunoreactivity, mutational status, and clinical behavior of PPGLs. Evaluation of SSTR-based therapies in metastatic PPGLs. METHODS: Retrospective analysis of a multicenter cohort of PPGLs at 6 specialized Endocrine Tumor Centers in Germany, The Netherlands, and Switzerland. Patients with PPGLs participating in the ENSAT registry were included. Clinical data were extracted from medical records, and immunohistochemistry (IHC) for SDHB and SSTR2 was performed in patients with available tumor tissue. Immunoreactivity of SSTR2 was investigated using Volante scores. The main outcome measure was the association of SSTR2 IHC positivity with genetic and clinical-pathological features of PPGLs. RESULTS: Of 202 patients with PPGLs, 50% were SSTR2 positive. SSTR2 positivity was significantly associated with SDHB- and SDHx-related PPGLs, with the strongest SSTR2 staining intensity in SDHB-related PPGLs (P = .01). Moreover, SSTR2 expression was significantly associated with metastatic disease independent of SDHB/SDHx mutation status (P < .001). In metastatic PPGLs, the disease control rate with first-line SSTR-based radionuclide therapy was 67% (n = 22, n = 11 SDHx), and with first-line "cold" somatostatin analogs 100% (n = 6, n = 3 SDHx). CONCLUSION: SSTR2 expression was independently associated with SDHB/SDHx mutations and metastatic disease. We confirm a high disease control rate of somatostatin receptor-based therapies in metastatic PPGLs.

Epigenetic drugs in somatostatin type 2 receptor radionuclide theranostics and radiation transcriptomics in mouse pheochromocytoma models.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are catecholamine-producing tumors. In inoperable and metastatic cases, somatostatin type 2 receptor (SSTR2) expression allows for peptide receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE. Insufficient receptor levels, however, limit treatment efficacy. This study evaluates whether the epigenetic drugs valproic acid (VPA) and 5-Aza-2'-deoxycytidine (DAC) modulate SSTR2 levels and sensitivity to [177Lu]Lu-DOTA-TATE in two mouse PCC models (MPC and MTT). Methods: Drug-effects on Sstr2/SSTR2 were investigated in terms of promoter methylation, mRNA and protein levels, and radiotracer binding. Radiotracer uptake was measured in subcutaneous allografts in mice using PET and SPECT imaging. Tumor growth and gene expression (RNAseq) were characterized after drug treatments. Results: DAC alone and in combination with VPA increased SSTR2 levels along with radiotracer uptake in vitro in MPC (high-SSTR2) and MTT cells (low-SSTR2). MTT but not MPC allografts responded to DAC and VPA combination with significantly elevated radiotracer uptake, although activity concentrations remained far below those in MPC tumors. In both models, combination of DAC, VPA and [177Lu]Lu-DOTA-TATE was associated with additive effects on tumor growth delay and specific transcriptional responses in gene sets involved in cancer and treatment resistance. Effects of epigenetic drugs were unrelated to CpG island methylation of the Sstr2 promoter. Conclusion: This study demonstrates that SSTR2 induction in mouse pheochromocytoma models has some therapeutic benefit that occurs via yet unknown mechanisms. Transcriptional changes in tumor allografts associated with epigenetic treatment and [177Lu]Lu-DOTA-TATE provide first insights into genetic responses of PCCs/PGLs, potentially useful for developing additional strategies to prevent tumor recurrence.

Case report: Incidentally discovered case of pheochromocytoma as a cause of long COVID-19 syndrome.

Pheochromocytomas (PCCs) are rare but potentially lethal tumors that arise from the adrenal medulla. The clinical suspicion and diagnosis of PCC can be challenging due to the non-specific nature of signs and symptoms. In many patients, infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could lead to long-term symptoms including fatigue, headaches, and cognitive dysfunction. Here, we present the case of a patient incidentally diagnosed with an adrenal mass that proved to be a PCC after imaging was performed due to persisting complaints after coronavirus disease 2019 (COVID-19) infection. A 37-year-old male patient was referred to our center because of a right-sided inhomogeneous adrenal mass, incidentally found during a computed tomographic scan of the thorax performed due to cough and dyspnea that persisted after COVID-19 infection. Other complaints that were present prior to COVID-19 infection included profuse sweating, dizziness, exhaustion with chronic fatigue, and concentration difficulties. The patient had no history of hypertension, his blood pressure was normal, and the 24-h ambulatory blood pressure monitoring confirmed normotension but with the absence of nocturnal dipping. Plasma normetanephrine was 5.7-fold above the upper limit (UL) of reference intervals (738 pg/ml, UL = 129 pg/ml), whereas plasma metanephrine and methoxytyramine were normal at 30 pg/ml (UL = 84 pg/ml) and <4 pg/ml (UL = 16 pg/ml), respectively. Preoperative preparation with phenoxybenzamine was initiated, and a 4-cm tumor was surgically resected. Profuse sweating as well as dizziness was resolved after adrenalectomy pointing toward PCC and not COVID-19-associated patient concerns. Altogether, this case illustrates the difficulties in recognizing the possibility of PCC due to the non-specific nature of signs and symptoms of the tumor, which in this case did not include hypertension and coincided with some of the symptoms of long COVID-19.

Personalized drug testing in human pheochromocytoma/paraganglioma primary cultures.

Aggressive pheochromocytomas and paragangliomas (PPGLs) are difficult to treat, and molecular targeting is being increasingly considered, but with variable results. This study investigates established and novel molecular-targeted drugs and chemotherapeutic agents for the treatment of PPGLs in human primary cultures and murine cell line spheroids. In PPGLs from 33 patients, including 7 metastatic PPGLs, we identified germline or somatic driver mutations in 79% of cases, allowing us to assess potential differences in drug responsivity between pseudohypoxia-associated cluster 1-related (n = 10) and kinase signaling-associated cluster 2-related (n = 14) PPGL primary cultures. Single anti-cancer drugs were either more effective in cluster 1 (cabozantinib, selpercatinib, and 5-FU) or similarly effective in both clusters (everolimus, sunitinib, alpelisib, trametinib, niraparib, entinostat, gemcitabine, AR-A014418, and high-dose zoledronic acid). High-dose estrogen and low-dose zoledronic acid were the only single substances more effective in cluster 2. Neither cluster 1- nor cluster 2-related patient primary cultures responded to HIF-2a inhibitors, temozolomide, dabrafenib, or octreotide. We showed particular efficacy of targeted combination treatments (cabozantinib/everolimus, alpelisib/everolimus, alpelisib/trametinib) in both clusters, with higher efficacy of some targeted combinations in cluster 2 and overall synergistic effects (cabozantinib/everolimus, alpelisib/trametinib) or synergistic effects in cluster 2 (alpelisib/everolimus). Cabozantinib/everolimus combination therapy, gemcitabine, and high-dose zoledronic acid appear to be promising treatment options with particularly high efficacy in SDHB-mutant and metastatic tumors. In conclusion, only minor differences regarding drug responsivity were found between cluster 1 and cluster 2: some single anti-cancer drugs were more effective in cluster 1 and some targeted combination treatments were more effective in cluster 2.

Native and Oxidized Low-Density Lipoproteins Increase the Expression of the LDL Receptor and the LOX-1 Receptor, Respectively, in Arterial Endothelial Cells.

Atherosclerotic artery disease is the major cause of death and an immense burden on healthcare systems worldwide. The formation of atherosclerotic plaques is promoted by high levels of low-density lipoproteins (LDL) in the blood, especially in the oxidized form. Circulating LDL is taken up by conventional and non-classical endothelial cell receptors and deposited in the vessel wall. The exact mechanism of LDL interaction with vascular endothelial cells is not fully understood. Moreover, it appears to depend on the type and location of the vessel affected and the receptor involved. Here, we analyze how native LDL (nLDL) and oxidized LDL (oxLDL) modulate the expression of their receptors-classical LDLR and alternative LOX-1-in endothelial cells derived from human umbilical artery (HUAECs), used as an example of a medium-sized vessel, which is typically affected by atherosclerosis. Exposure of HUAECs to nLDL resulted in moderate nLDL uptake and gradual increase in LDLR, but not LOX-1, expression over 24 h. Conversely, exposure of HUAECs to oxLDL, led to significant accumulation of oxLDL and rapid induction of LOX-1, but not LDLR, within 7 h. These activation processes were associated with phosphorylation of protein kinases ERK1/2 and p38, followed by activation of the transcription factor AP-1 and its binding to the promoters of the respective receptor genes. Both nLDL-induced LDLR mRNA expression and oxLDL-induced LOX-1 mRNA expression were abolished by blocking ERK1/2, p-38 or AP-1. In addition, oxLDL, but not nLDL, was capable of inducing LOX-1 through the NF-κB-controlled pathway. These observations indicate that in arterial endothelial cells nLDL and oxLDL signal mainly via LDLR and LOX-1 receptors, respectively, and engage ERK1/2 and p38 kinases, and AP-1, as well as NF-κB transcription factors to exert feed-forward regulation and increase the expression of these receptors, which may perpetuate endothelial dysfunction in atherosclerosis.

Effects of digitalized university curriculum-associated teaching on the equilibrium of autonomic neurophysiology and disposition of learners in medical school (EDUCATE-AND-LEARN): protocol for a randomized crossover study.

BACKGROUND: Homoeostasis of the autonomic nervous system (ANS) contributes to cognitive functional integrity in learners and can be greatly influenced by emotions and stress. While moderate stress can enhance learning and memory processes, long-term stress compromises learning performance in a face-to-face classroom environment. Integrative online learning and communication tools were shown to be beneficial for visualization and comprehension but their effects on the ANS are poorly understood. We aim to assess the effects of video conference-supported live lectures compared to on-site classroom teaching on autonomic functions and their association with learning performance. METHODS AND DESIGN: Fifty mentally and physically healthy medical students will be enrolled in a randomized two-period crossover study. Subjects will attend a seminar, which is held in face-to-face and simultaneously transmitted via videoconference. Subjects will be allocated in two arms in a randomized sequence determining the order in which both seminar settings will be attended. At baseline and throughout the interactive seminar subjects will undergo detailed autonomic testing comprising neurocardiac (heart rate variability), sudomotor (sympathetic skin response), neurovascular (laser Doppler flowmetry) and pupillomotor (pupillography) function. Furthermore, learning progress will be evaluated using pre- and post-tests on the seminar subject and emotions will be assessed using profile of mood state (POMS) questionnaire. STATISTICAL ANALYSIS: Carryover effects will be handled using a two-way repeated measures (mixed model). Between-group differences (baseline vs face-to-face vs videoconference) will be determined using one-way analysis of variance ANOVA followed by Student-Newman-Keul test. LIMITATIONS AND STRENGTHS: This study may elucidate complex interactions between autonomic and emotional dynamics during conventional on-site and video conference-based teaching, thus providing a basis for customized learning and teaching methods. Understanding and utilizing advanced distance learning strategies is particularly important during the current pandemic, which has been limiting on-site teaching dramatically in nearly all countries of the world.

HIF2alpha-Associated Pseudohypoxia Promotes Radioresistance in Pheochromocytoma: Insights from 3D Models.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are rare neuroendocrine tumors arising from chromaffin tissue located in the adrenal or ganglia of the sympathetic or parasympathetic nervous system. The treatment of non-resectable or metastatic PCCs/PGLs is still limited to palliative measures, including somatostatin type 2 receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE as one of the most effective approaches to date. Nevertheless, the metabolic and molecular determinants of radiation response in PCCs/PGLs have not yet been characterized. This study investigates the effects of hypoxia-inducible factor 2 alpha (HIF2α) on the susceptibility of PCCs/PGLs to radiation treatments using spheroids grown from genetically engineered mouse pheochromocytoma (MPC) cells. The expression of Hif2α was associated with the significantly increased resistance of MPC spheroids to external X-ray irradiation and exposure to beta particle-emitting [177Lu]LuCl3 compared to Hif2α-deficient controls. Exposure to [177Lu]LuCl3 provided an increased long-term control of MPC spheroids compared to single-dose external X-ray irradiation. This study provides the first experimental evidence that HIF2α-associated pseudohypoxia contributes to a radioresistant phenotype of PCCs/PGLs. Furthermore, the external irradiation and [177Lu]LuCl3 exposure of MPC spheroids provide surrogate models for radiation treatments to further investigate the metabolic and molecular determinants of radiation responses in PCCs/PGLs and evaluate the effects of neo-adjuvant-in particular, radiosensitizing-treatments in combination with targeted radionuclide therapies.

New Horizons: Novel Adrenal Regenerative Therapies.

Adrenal insufficiency requires lifelong corticoid replacement therapies. However, current therapies are not able to replace the physiological circadian pattern of the adrenal cortex and are associated with many metabolic, vascular, neuroendocrine, and mental perturbations. Therefore, regenerative and more curative strategies would be desirable. In the current perspective, we describe emerging new regenerative therapies for the treatment of adrenal insufficiency. In particular, we discuss gene therapy and cell replacement strategies. Furthermore, we discuss how adrenal cells might be used as a source for regenerative therapies of nonadrenal neurodegenerative diseases such as Parkinson disease.

Current Management of Pheochromocytoma/Paraganglioma: A Guide for the Practicing Clinician in the Era of Precision Medicine.

Pheochromocytomas and paragangliomas (PCC/PGLs) are rare, mostly catecholamine-producing neuroendocrine tumors of the adrenal gland (PCCs) or the extra-adrenal paraganglia (PGL). They can be separated into three different molecular clusters depending on their underlying gene mutations in any of the at least 20 known susceptibility genes: The pseudohypoxia-associated cluster 1, the kinase signaling-associated cluster 2, and the Wnt signaling-associated cluster 3. In addition to tumor size, location (adrenal vs. extra-adrenal), multiplicity, age of first diagnosis, and presence of metastatic disease (including tumor burden), other decisive factors for best clinical management of PCC/PGL include the underlying germline mutation. The above factors can impact the choice of different biomarkers and imaging modalities for PCC/PGL diagnosis, as well as screening for other neoplasms, staging, follow-up, and therapy options. This review provides a guide for practicing clinicians summarizing current management of PCC/PGL according to tumor size, location, age of first diagnosis, presence of metastases, and especially underlying mutations in the era of precision medicine.

Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

Synergistic Highly Potent Targeted Drug Combinations in Different Pheochromocytoma Models Including Human Tumor Cultures.

There are no officially approved therapies for metastatic pheochromocytomas apart from ultratrace 131I-metaiodbenzylguanidine therapy, which is approved only in the United States. We have, therefore, investigated the antitumor potential of molecular-targeted approaches in murine pheochromocytoma cell lines [monocyte chemoattractant protein (MPC)/monocyte chemoattractant protein/3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], immortalized mouse chromaffin Sdhb-/- cells, three-dimensional pheochromocytoma tumor models (MPC/MTT spheroids), and human pheochromocytoma primary cultures. We identified the specific phosphatidylinositol-3-kinase α inhibitor BYL719 and the mammalian target of rapamycin inhibitor everolimus as the most effective combination in all models. Single treatment with clinically relevant doses of BYL719 and everolimus significantly decreased MPC/MTT and Sdhb-/- cell viability. A targeted combination of both inhibitors synergistically reduced MPC and Sdhb-/- cell viability and showed an additive effect on MTT cells. In MPC/MTT spheroids, treatment with clinically relevant doses of BYL719 alone or in combination with everolimus was highly effective, leading to a significant shrinkage or even a complete collapse of the spheroids. We confirmed the synergism of clinically relevant doses of BYL719 plus everolimus in human pheochromocytoma primary cultures of individual patient tumors with BYL719 attenuating everolimus-induced AKT activation. We have thus established a method to assess molecular-targeted therapies in human pheochromocytoma cultures and identified a highly effective combination therapy. Our data pave the way to customized combination therapy to target individual patient tumors.

Targeting Cyclooxygenase-2 in Pheochromocytoma and Paraganglioma: Focus on Genetic Background.

Cyclooxygenase 2 (COX-2) is a key enzyme of the tumorigenesis-inflammation interface and can be induced by hypoxia. A pseudohypoxic transcriptional signature characterizes pheochromocytomas and paragangliomas (PPGLs) of the cluster I, mainly represented by tumors with mutations in von Hippel-Lindau (VHL), endothelial PAS domain-containing protein 1 (EPAS1), or succinate dehydrogenase (SDH) subunit genes. The aim of this study was to investigate a possible association between underlying tumor driver mutations and COX-2 in PPGLs. COX-2 gene expression and immunoreactivity were examined in clinical specimens with documented mutations, as well as in spheroids and allografts derived from mouse pheochromocytoma (MPC) cells. COX-2 in vivo imaging was performed in allograft mice. We observed significantly higher COX-2 expression in cluster I, especially in VHL-mutant PPGLs, however, no specific association between COX-2 mRNA levels and a hypoxia-related transcriptional signature was found. COX-2 immunoreactivity was present in about 60% of clinical specimens as well as in MPC spheroids and allografts. A selective COX-2 tracer specifically accumulated in MPC allografts. This study demonstrates that, although pseudohypoxia is not the major determinant for high COX-2 levels in PPGLs, COX-2 is a relevant molecular target. This potentially allows for employing selective COX-2 inhibitors as targeted chemotherapeutic agents and radiosensitizers. Moreover, available models are suitable for preclinical testing of these treatments.

Strain-specific metastatic phenotypes in pheochromocytoma allograft mice

Somatostatin receptor-targeting endoradiotherapy offers potential for treating metastatic pheochromocytomas and paragangliomas, an approach likely to benefit from combination radiosensitization therapy. To provide reliable preclinical in vivo models of metastatic disease, this study characterized the metastatic spread of luciferase-expressing mouse pheochromocytoma (MPC) cells in mouse strains with different immunologic conditions. Bioluminescence imaging showed that, in contrast to subcutaneous non-metastatic engraftment of luciferase-expressing MPC cells in NMRI-nude mice, intravenous cell injection provided only suboptimal metastatic spread in both NMRI-nude mice and hairless SCID (SHO) mice. Treatment of NMRI-nude mice with anti-Asialo GM1 serum enhanced metastatic spread due to substantial depletion of natural killer (NK) cells. However, reproducible metastatic spread was only observed in NK cell-defective SCID/beige mice and in hairless immunocompetent SKH1 mice bearing disseminated or liver metastases, respectively. Liquid chromatography tandem mass spectrometry of urine samples showed that subcutaneous and metastasized tumor models exhibit comparable renal monoamine excretion profiles characterized by increasing urinary dopamine, 3-methoxytyramine, norepinephrine and normetanephrine. Metastases-related epinephrine and metanephrine were only detectable in SCID/beige mice. Positron emission tomography and immunohistochemistry revealed that all metastases maintained somatostatin receptor-specific radiotracer uptake and immunoreactivity, respectively. In conclusion, we demonstrate that intravenous injection of luciferase-expressing MPC cells into SCID/beige and SKH1 mice provides reproducible and clinically relevant spread of catecholamine-producing and somatostatin receptor-positive metastases. These standardized preclinical models allow for precise monitoring of disease progression and should facilitate further investigations on theranostic approaches against metastatic pheochromocytomas and paragangliomas.

Anti-Tumorigenic and Anti-Metastatic Activity of the Sponge-Derived Marine Drugs Aeroplysinin-1 and Isofistularin-3 against Pheochromocytoma In Vitro.

Over 10% of pheochromocytoma and paraganglioma (PPGL) patients have malignant disease at their first presentation in the clinic. Development of malignancy and the underlying molecular pathways in PPGLs are poorly understood and efficient treatment strategies are missing. Marine sponges provide a natural source of promising anti-tumorigenic and anti-metastatic agents. We evaluate the anti-tumorigenic and anti-metastatic potential of Aeroplysinin-1 and Isofistularin-3, two secondary metabolites isolated from the marine sponge Aplysina aerophoba, on pheochromocytoma cells. Aeroplysinin-1 diminished the number of proliferating cells and reduced spheroid growth significantly. Beside these anti-tumorigenic activity, Aeroplysinin-1 decreased the migration ability of the cells significantly (p = 0.01), whereas, the invasion capacity was not affected. Aeroplysinin-1 diminished the high adhesion capacity of the MTT cells to collagen (p < 0.001) and, furthermore, reduced the ability to form spheroids significantly. Western Blot and qRT-PCR analysis showed a downregulation of integrin ß1 that might explain the lower adhesion and migration capacity after Aeroplysinin-1 treatment. Isofistularin-3 showed only a negligible influence on proliferative and pro-metastatic cell properties. These in vitro investigations show promise for the application of the sponge-derived marine drug, Aeroplysinin-1 as anti-tumorigenic and anti-metastatic agent against PPGLs for the first time.

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells.

Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.

Impact of Aldosterone Synthase Inhibitor FAD286 on Steroid Hormone Profile in Human Adrenocortical Cells.

Inhibition of aldosterone synthase (CYP11B2) is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone effects. FAD286 is the best characterized aldosterone synthase inhibitor. However, to date, no study has used sensitive liquid chromatography-tandem mass spectrometry to characterize in detail the effect of FAD286 on the secreted steroid hormone profile of adrenocortical cells. Basal aldosterone production in NCI-H295R cells was detectable and 9-fold elevated after stimulation with angiotensin II. FAD286 inhibited this increase, showing a maximal effect at 10 nmol/l. Higher concentrations of FAD286 did not further reduce aldosterone concentrations, but showed a parallel reduction in corticosterone, cortisol and cortisone levels, reflecting additional inhibition of steroid-11ß-hydroxylase (CYP11B1). Pregnenolone, progesterone and 17-OH-progesterone levels remained unaffected. In conclusion, the aldosterone synthase inhibitor FAD286 lowers angiotensin II-induced aldosterone concentrations in adrenocortical cells but the relative lack of selectivity over CYP11B1 is evident at higher FAD286 concentrations.