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Phaeochromocytomas and paragangliomas are rare neuroendocrine tumours. So far, over 20 causative genes have been identified, of which the most frequent and strongest indicator for malignancies are mutations in succinate dehydrogenase subunit B. No curative therapy is available for patients with metastases resulting in poor prognosis. Therapy development has been hindered by lack of suitable model systems. The succinate dehydrogenase complex is located in the inner membrane of the mitochondria and plays a crucial role in the oxidative phosphorylation chain and the tricarboxylic acid-cycle. Succinate dehydrogenase deficiency results in accumulation of the oncometabolite succinate inducing hypoxia inducible factor stabilization, deoxyribonucleic acid and histone methylation inhibition, and impaired production of adenosine triphosphate. It remains unknown which combination of pathways and/or triggers are decisive for metastases development. In this review, the role of mitochondria in malignant succinate dehydrogenase subunit B-associated phaeochromocytomas and paragangliomas and implications for mitochondria as therapeutic target are discussed., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Pheochromocytomas/paragangliomas (PHEOs/PGLs) are rare in children with only a few SDHB mutation-related cases. Previous studies on children were conducted in small cohorts. This large set of pediatric patients provides robust data in the evaluation of clinical outcomes., Methods: Sixty-four pediatric PHEO/PGL patients with SDHB germline mutations were included in the present study. The clinical presentation, disease course, and survival rate were evaluated., Results: Thirty-eight males and 26 females were diagnosed with PHEO/PGL at a median age of 13 years. The majority of patients displayed norepinephrine hypersecretion and 73.44% initially presented with a solitary tumor. Metastases developed in 70% of patients at the median age of 16 years and were mostly diagnosed first 2 years and in years 12-18 post-diagnosis. The presence of metastases at the time of diagnosis had a strong negative impact on survival in males but not in females. The estimated 5-, 10-, and 20-year survival rates were 100%, 97.14%, and 77.71%, respectively., Conclusion: The present report has highlighted several important aspects in the management of pediatric patients with SDHB mutations associated-PHEO/PGL. Initial diagnostic evaluation of SDHB mutation carriers should be started at age of 5-6 years with initial work-up focusing on abdominal region. Thorough follow-up is crucial first 2 years post-diagnosis and more frequent follow-ups are needed in years 10-20 post-diagnosis due to the increased risk of metastases. Although this age group developed metastasis as early as 5 years from diagnosis, we have shown that the overall 20-year prognosis and survival are good.

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Purpose: Phosphoinositide-dependent kinase 1 (PDK1) is highly expressed in many solid tumors. And several studies have demonstrated that PDK1 has been an emerging and promising target for anti-cancer therapies. However, the role of PDK1 has not been studied so far in malignant pheochromocytoma (PCC)., Methods: In this study, immunohistochemical staining was performed to investigate the protein level of PDK1 in 63 PCC tissue samples, of which 49 were benign and 14 were malignant. In addition, we evaluated the effect of inhibition of PDK1 with siRNA on cell growth, apoptosis and invasive capacity in PC12 cells and identified the underlying mechanisms., Results: We found that PDK1 was overexpressed in malignant PCC tissues, and knockdown of PDK1 with siRNA significantly inhibited cell proliferation, increased apoptosis induction, and attenuated cell migration and invasive capacity in PC12 cells. We also showed that knockdown of PDK1 significantly reduced the phosphorylation of Akt at threonine 308 (p-Akt T308) but did not alter the serine phosphorylation of Akt on the S473 site (p-Akt S473). Furthermore, we found that the p-Akt expression was noticeably decreased after knockdown of PDK1, but the t-Akt expression did not show a significant decrease., Conclusion: We have demonstrated for the first time that PDK1 is overexpressed in human malignant PCC and plays an important role in the malignant biological behaviors of PC12 cell. Specifically, we have revealed that knockdown of PDK1 could attenuate activation of the Akt signaling. These data suggest that PDK1 could be a new promising potential therapeutic target in human cancer treatment for malignant PCC.

Pheochromocytoma (PC) and paraganglioma (PGL) are rare neuroendocrine tumors that occur throughout the body from the base of the skull to the pelvis. Sympathetic catecholamine-secreting tumors may be associated with hyperadrenergic symptoms and long-term morbidity if they are untreated. Typically biochemically silent, head and neck PGLs may result in cranial nerve palsies and symptoms due to localized mass effect. Tumors can arise sporadically or as part of an inheritable PC-PGL syndrome. Up to 40% of tumors are recognized to be associated with germline mutations in an increasing array of susceptibility genes, including those that appear to arise sporadically. Most commonly, up to 25% of all PC-PGLs are associated with mutations in one of the succinate dehydrogenase (SDH) enzyme subunit genes. The resulting familial PC-PGL syndrome varies according to the affected enzyme subunit (most commonly SDHB and SDHD mutations) with respect to tumor prevalence, location, age of onset, and risk of malignancy. Patients with SDH enzyme mutations have increased lifetime risk of developing multifocal tumors and malignancy. Early recognition of individuals at high risk, genetic testing, screening of family members, and lifelong surveillance programs are recommended, but not without health, economic, and psychologic implications. Anatomic and functional imaging is key to diagnosis, staging, treatment planning, and lifelong surveillance of these individuals. Radiologists must be aware of the imaging appearance of these varied tumors. © RSNA, 2019.

Humans are constantly exposed to a multitude of environmental chemicals that may disturb endocrine functions. It is crucial to identify such chemicals and uncover their mode-of-action to avoid adverse health effects. 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) catalyze the formation of cortisol and aldosterone, respectively, in the adrenal cortex. Disruption of their synthesis by exogenous chemicals can contribute to cardio-metabolic diseases, chronic kidney disease, osteoporosis, and immune-related disorders. This study applied in silico screening and in vitro evaluation for the discovery of xenobiotics inhibiting CYP11B1 and CYP11B2. Several databases comprising environmentally relevant pollutants, chemicals in body care products, food additives and drugs were virtually screened using CYP11B1 and CYP11B2 pharmacophore models. A first round of biological testing used hamster cells overexpressing human CYP11B1 or CYP11B2 to analyze 25 selected virtual hits. Three compounds inhibited CYP11B1 and CYP11B2 with IC 50 values below 3 μM. The most potent inhibitor was epoxiconazole (IC 50 value of 623 nM for CYP11B1 and 113 nM for CYP11B2, respectively); flurprimidol and ancymidol were moderate inhibitors. In a second round, these three compounds were tested in human adrenal H295R cells endogenously expressing CYP11B1 and CYP11B2, confirming the potent inhibition by epoxiconazole and the more moderate effects by flurprimidol and ancymidol. Thus, the in silico screening, prioritization of chemicals for initial biological tests and use of H295R cells to provide initial mechanistic information is a promising strategy to identify potential endocrine disruptors inhibiting corticosteroid synthesis. A critical assessment of human exposure levels and in vivo evaluation of potential corticosteroid disrupting effects by epoxiconazole is required., (Copyright © 2019. Published by Elsevier Ltd.)

The mitochondrial enzyme succinate dehydrogenase (SDH) acts as a tumor suppressor. Biallelic inactivation of one of the genes encoding for SDH subunits (collectively named SDHx) leads to complete loss of the protein function and the development of diverse group of tumors. Pheochromocytomas-paragangliomas are the prime example of hereditary tumors caused by SDH deficiency. In this review, we discuss the roles of imaging examinations, and illustrate new insights into genotype-imaging phenotype relationships., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

In chromaffin cells, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) are mainly involved in catecholamine synthesis. In this study, we evaluated the association between the status of catecholamine-synthesizing enzymes and histopathological features of pheochromocytoma and extraadrenal paraganglioma with special emphasis upon their postoperative clinical behavior. Immunohistochemical evaluation of TH, DBH, AADC, PNMT, Ki 67, and S-100 was performed in 29 pheochromocytoma and 10 extraadrenal paraganglioma and one lymph node harboring metastatic pheochromocytoma. Among these cases, metastasis was subsequently developed in three cases. Urinary normetanephrine (U-NM) levels were significantly higher in clinical metastatic cases than non-metastatic ones. Ki 67 labeling index was significantly higher in both clinical metastatic cases and the Adrenal Gland Scaled Score (PASS) score of ≧ 4 cases than PASS < 4 cases, although this score was originally used in pheochromocytoma. H-score of AADC and DBH were significantly lower in PASS ≧ 4 cases than those with < 4 cases, and in the cases associated with intratumoral necrosis (n = 4), the presence of spindle shaped tumor cells (n = 4), and large nests of cells or diffuse growth (n = 5). Lower status of intratumoral AADC could be related to poor differentiation of tumor cells in both catecholamine production and morphology and could be related to aggressive biological behavior of both pheochromocytoma and extraadrenal paraganglioma.

Hypoxia is a primary factor in many pathological conditions. Hypoxic cell death is commonly attributed to metabolic failure and oxidative injury. cAMP-dependent protein kinase A (PKA) is activated in hypoxia and regulates multiple enzymes of the mitochondrial electron transport chain, thus may be implicated in cellular energy depletion and hypoxia-induced cell death. Wild type (WT) PC-12 cells and PKA activity-deficient 123.7 PC-12 cells were exposed to 3, 6, 12 and 24h hypoxia (0.1% or 5% O 2 ). Hypoxia, at 24h 0.1% O 2 , induced cell death and increased reactive oxygen species (ROS) in WT PC-12 cells. Despite lower ATP levels in normoxic 123.7 cells than in WT cells, hypoxia only decreased ATP levels in WT cells. However, menadione-induced oxidative stress similarly affected both cell types. While mitochondrial COX IV expression remained consistently higher in 123.7 cells, hypoxia decreased COX IV expression in both cell types. N-acetyl cysteine antioxidant treatment blocked hypoxia-induced WT cell death without preventing ATP depletion. Transient PKA catα expression in 123.7 cells partially restored hypoxia-induced ROS but did not alter ATP levels or COX IV expression. We conclude that PKA signaling contributes to hypoxic injury, by regulating oxidative stress rather than by depleting ATP levels. Therapeutic strategies targeting PKA signaling may improve cellular adaptation and recovery in hypoxic pathologies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ganglioneuromas represent the most well-differentiated spectrum of neoplasia arising from the sympathetic nervous system, while neuroblastomas represent the most poorly differentiated counterpart, and ganglioneuroblastomas represent intermediate stages of differentiation. Small series of cases have documented the co-occurrence of ganglioneuroma with a pheochromocytoma (Pheo)/paraganglioma (PGL) component. We report the clinicopathologic features of eight such cases, diagnosed between 2003 and 2015 with a mean follow-up of 22 months (1-47), which were evaluated for syndrome associations, SDHB expression, and clinical outcome. Mutations of the succinate dehydrogenase (SDH) complex subunits (A, B, C, D, and SDHAF2) have been implicated in predicting metastatic behavior and in identifying possible paraganglioma syndromes. The proliferative index was calculated by manual quantification of Ki-67-positive cells at selected hot-spots using ImageJ (NIH). In our series, composite Pheo/PGL-ganglioneuromas predominantly involved the adrenal gland (Pheo 7, PGL 1). The cases had an equal gender distribution (males 4, females 4), with a mean age at diagnosis of 67 years (range 53 to 86 years), an average size of 5.2 cm (range 2 to 8.2 cm), an average weight of 49.3 g (7.8 to 144.7 g, n = 6), and the majority were functionally active (7 of 8, 88%). The mean Ki67 proliferation rate was 2% (range 0.3 to 3%), and all cases retained SDHB expression (8/8, 100%). No patient (0/8, 0%) developed metastatic disease on follow-up. One patient had a retroperitoneal composite PGL-ganglioneuroma in the setting of neurofibromatosis type 1. No recurrent disease or other associations were identified. In our study, composite Pheo/PGL-ganglioneuromas predominantly affected the adrenal gland in older patients, showed no loss of SDHB, and no disease recurrence was identified.

MicroRNAs (miRNAs) play an important role in multiple biological processes, and many miRNAs have been shown to regulate cell proliferation and apoptosis. In this study, we investigated the role of miR-15b-5p in cell proliferation and apoptosis in PC12 cells. We found that overexpression of miR-15b-5p could decrease cell proliferation and induce apoptosis and cytotoxic activities in PC12 cells. Bioinformatics analysis and luciferase activities assays showed that miR-15b-5p might target extracellular signal-regulated kinase 1 (ERK1) by binding to its 3'-untranslated region (3'-UTR). Moreover, we also found that overexpression of ERK1 could attenuate the effects of miR-15b-5p in PC12 cells. Finally, our results suggest that miR-15b-5p might inhibit cell proliferation and induce apoptosis in PC12 cells by targeting ERK1., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Oxidative stress has been implicated in the pathogenesis of many kinds of neurodegenerative disorders, particularly Parkinson's disease. Quercetin is a bioflavonoid found ubiquitously in fruits and vegetables, and has antioxidative activity. However, the underlying mechanism of the antioxidative effect of quercetin in neurodegenerative diseases has not been well explored. Here, we investigated the antioxidative effect and underlying molecular mechanisms of quercetin on PC-12 cells. We found that PC-12 cells pretreated with quercetin exhibited an increased cell viability and reduced lactate dehydrogenase (LDH) release when exposed to hydrogen peroxide (H₂O₂). The significantly-alleviated intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and lipoperoxidation of the cell membrane of PC-12 cells induced by H₂O₂ were observed in the quercetin pretreated group. Furthermore, quercetin pretreatment markedly reduced the apoptosis of PC-12 cells and hippocampal neurons. The inductions of antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in PC-12 cells exposed to H₂O₂ were significantly reduced by preatment with quercetin. In addition, quercetin pretreatment significantly increased Bcl-2 expression, and reduced Bax, cleaved caspase-3 and p53 expressions. In conclusion, this study demonstrated that quercetin exhibited a protective effect against oxidative stress-induced apoptosis in PC-12 cells. Our findings suggested that quercetin may be developed as a novel therapeutic agent for neurodegenerative diseases induced by oxidative stress., Competing Interests: The authors declare no conflict of interest.

This study aims to examine the expression profiles of the miR-183 cluster (miR-96/182/183) in pheochromocytoma. Pheochromocytoma tissues were prospectively collected from 50 patients with pheochromocytoma. Expression of miR-183 cluster members and SDHB protein expression were analyzed in these tissues by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. The expression of miR-183 cluster members in pheochromocytomas was correlated with the clinical and pathological parameters of these patients. The expression levels of miR-183 cluster members were predominantly downregulated or deleted in pheochromocytoma. Low expression or deletion of miR-96 was predominantly noted in younger patients with pheochromocytoma (<50 years, P=.01). Female patients in the study group showed marked deletion of miR-182 (P=.05). Deletion of the cluster was also associated with SDHB protein expression in pheochromocytoma. Moreover, patients with low miR-183 cluster expression had a slightly better survival rate when compared with patients with high expression. To conclude, the findings indicate a role for miR-183 cluster members in the pathogenesis and clinical progression of pheochromocytoma., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hepatoid adenocarcinoma is a rare extrahepatic tumor, which shows morphological and immunohistochemical similarities to hepatocellular carcinoma (HCC). Hence, hepatoid adenocarcinoma can cause diagnostic confusion with HCC. Arginase-1 immunostain has been recently shown to be an excellent marker of normal hepatocytes and is a sensitive and specific marker for HCC. However, the expression of Arginase-1 in hepatoid adenocarcinoma has not been evaluated in detail. Eight cases of hepatoid adenocarcinoma were immunostained with Arginase-1, Hepar-1, Glypican-3, CK7, CK20, CK19, polyclonal carcinoembryonic antigen, ɑ-fetoprotein, CDX2, and TTF-1. Albumin in situ hybridization was performed in 4 cases. All 8 cases were positive for Hepar-1. Arginase-1 was positive in 5 (62.5%) of 8 cases; 2 of these cases showed diffuse staining, while 3 showed patchy staining. Glypican-3, CK7 and ɑ-fetoprotein were each positive in 4 (50%) of 8 cases. CK19 was positive in 3 (37.5%) of 8 cases. polyclonal carcinoembryonic antigen showed canalicular staining in 3 (37.5%) of 8 cases and albumin in situ hybridization was positive in 3 (75%) of 4 cases. CDX2 was positive in 2 (25%) of 8 cases, both arising from the stomach. CK20 was positive in 1 (12.5%) of 8 case while TTF-1 was negative in all cases. Hepatoid adenocarcinoma has a similar immunostaining profile as HCC. Arginase-1 expression is common (62.5%) in hepatoid adenocarcinoma and hence it is not useful in distinguishing HCC from hepatoid adenocarcinoma., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Objective: Multiple Endocrine Neoplasia type 2 (MEN2) is a rare genetic disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and primary hyperparathyroidism. MEN2 is an autosomal dominant syndrome caused by mutations in the RET proto-oncogene. In the vast majority of patients, the mutations are localized in exons 10, 11 and 13-15 of the RET gene. Rare variants located in exon 8 were recently identified but their clinical significance remains unclear., Design and Methods: We studied two sisters presenting with pheochromocytoma as the first tumor. One of the sisters was diagnosed with a right pheochromocytoma at the age of 44 and at age 53 she developed an invasive left pheochromocytoma with no other endocrine neoplasia. The other sister was diagnosed with a left pheochromocytoma at age 50 and at age 64 she had a right phemochromocytoma and MTC. Neither of the two sisters presented evidence of primary hyperparathyroidism. Mutations of the RET proto-oncogene were investigated by DNA sequencing., Results: We detected a germline missense variant in RET exon 8 (p.Cys531Arg) in both sisters. The p.Cys531Arg variant was not present in a third 50-year-old sister who has remained to date clinically unaffected., Conclusion: This is the first case showing the p.Cys531Arg variant in RET exon 8 co-segregating with family members affected by a syndrome reminiscent of MEN2A. Our results suggest that this variant has a specific genotype-phenotype correlation as it is associated with the development of pheochromocytoma before the onset of MTC.

Malignant pheochromocytoma/paraganglioma (PCC/PGL) is defined by the presence of metastases at non-chromaffin sites, which makes it difficult to prospectively diagnose malignancy. Here, we performed array CGH (aCGH) and paired gene expression profiling of fresh, frozen PCC/PGL samples (n = 12), including three malignant tumors, to identify genes that distinguish benign from malignant tumors. Most PCC/PGL cases showed few copy number aberrations, regardless of malignancy status, but mRNA analysis revealed that 390 genes were differentially expressed in benign and malignant tumors. Expression of the enzyme, phenylethanolamine N-methyltransferase (PNMT), which catalyzes the methylation of norepinephrine to epinephrine, was significantly lower in malignant PCC/PGL as compared to benign samples. In 62 additional samples, we confirmed that PNMT mRNA and protein levels were decreased in malignant PCC/PGL using quantitative real-time polymerase chain reaction and immunohistochemistry. The present study demonstrates that PNMT downregulation correlates with malignancy in PCC/PGL and identifies PNMT as one of the most differentially expressed genes between malignant and benign tumors., Competing Interests: The authors have no conflicts of interest to disclose.

Germline mutations occur in up to 30-40% of pheochromocytoma/paraganglioma, with mutations in the succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD) being the most common. Blood samples are favored for obtaining high quality DNA, however, leukocytes can also be obtained by collecting saliva. The aim of this study was to determine whether SDHB and SDHD gene mutations in patients with pheochromocytoma/paraganglioma could be determined using a salivary sample. Paired blood and salivary samples were collected from 30 patients: 9 SDHB mutation positive, 13 with a SDHD mutation, and 8 without any SDHx mutations. The Oragene DISCOVER kit was used to collect and extract DNA from saliva. Blood DNA was extracted from EDTA blood samples. The DNA purification and concentration were measured by spectrophotometry. The 8 exons of SDHB and the 4 exons of SDHD were amplified and sequenced by PCR-based bidirectional Sanger sequencing. Total DNA yields from blood DNA were similar to those obtained from saliva DNA [mean (±SD) saliva vs. blood DNA concentration 514.6 (±580.8) ng/µl vs. 360.9 (±262.7) ng/µl; p=0.2)]. The purity of the saliva DNA samples was lower than that of blood [mean OD260/OD280 ratio 1.78 (±0.13) vs. 1.87 (±0.04); p=0.001, respectively], indicating more protein contamination in the saliva-extracted DNA. This study shows that salivary DNA collected from patients with pheochromocytoma/paraganglioma is a good alternative for extraction of genomic DNA for its high DNA concentration and acceptable purity and can be used as an alternative to blood derived DNA in screening for SDHB and SDHD mutations., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Intersecting a genome-wide expression profile of metastatic and nonmetastatic human neuroblastoma xenograft variants with expression profiles of tumours from stage 1 and 4 neuroblastoma patients, we previously characterised hexokinase 2 (HK2) as a gene whose expression was upregulated in both metastatic neuroblastoma variants and tumours from stage 4 neuroblastoma patients., Methods: Local and metastatic neuroblastoma cell variants as well as metastatic neuroblastoma cells genetically manipulated to downregulate the expression of HK2 were utilised for in vitro and in vivo examinations of the involvement of HK2 in neuroblastoma., Results: Hexokinase 2 expression and its activity levels were increased in neuroblastoma metastatic variants as compared with the local variants. The upregulation of HK2 confers upon the metastatic cells high resistance to the antiproliferative effect of the HK2 inhibitor 3-BrPa and to the chemotherapy agent Deferoxamine. The inhibition of HK2 transcript lowered the proliferation and motility of sh-HK2 cells as compared with sh-control cells. Mice that were inoculated with sh-HK2 cells had a lower incidence of local tumours, smaller tumour volumes and a diminished load of lung metastasis compared with mice inoculated with sh-control cells., Conclusions: Hexokinase 2 plays a significant role in shaping the malignant phenotype of neuroblastoma and influences the progression of this disease.

Context: Although adrenal vein sampling is the standard method to distinguish unilateral from bilateral forms of primary aldosteronism, it is an invasive and technically difficult procedure. (11)C-metomidate (MTO)-positron emission tomography was reported as a potential replacement for adrenal vein sampling. However, MTO has low selectivity for CYP11B2 over CYP11B1., Objective: This study aimed to determine the selectivity of (18)F-CDP2230, a new imaging agent, for CYP11B2 over CYP11B1 and determine whether the biodistribution profile of (18)F-CDP2230 is favorable for imaging CYP11B2., Methods: The IC50 of CDP2230 for the enzymatic activities of CYP11B2 and CYP11B1 was determined using cells with stable expression of either enzyme. In vitro autoradiography of human adrenal sections with aldosterone-producing adenomas was performed to confirm the specific binding ability of (18)F-CDP2230 to CYP11B2-expressing regions. Furthermore, positron emission tomography and magnetic resonance imaging were performed to evaluate the biodistribution of (18)F-CDP2230 in rats., Results: Although CDP2230 showed a significantly lower affinity for CYP11B2 and CYP11B1 than did MTO analogues, its selectivity for CYP11B2 over CYP11B1 was higher than that of MTO analogues. In vitro autoradiography revealed that the binding of (18)F-CDP2230 to CYP11B2-expressing regions in the adrenal gland was more specific than that of (123)I-IMTO. Moreover, the biodistribution study showed that (18)F-CDP2230 accumulated in adrenal glands with low background uptake., Conclusions: Our study showed a high selectivity of (18)F-CDP2230 for CYP11B2 over CYP11B1 with a favorable biodistribution for imaging CYP11B2. (18)F-CDP2230 is a promising imaging agent for detecting unilateral subtypes of primary aldosteronism.

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes and tumor suppressors. Loss-of-function mutations give rise to hereditary paragangliomas/pheochromocytomas and hereditary leiomyomatosis and renal cell carcinoma. Inactivation of SDH and FH results in an abnormal accumulation of their substrates succinate and fumarate, leading to inhibition of numerous α-ketoglutarate dependent dioxygenases, including histone demethylases and the ten-eleven-translocation (TET) family of 5-methylcytosine (5 mC) hydroxylases. To evaluate the distribution of DNA and histone methylation, we used immunohistochemistry to analyze the expression of 5 mC, 5-hydroxymethylcytosine (5 hmC), TET1, H3K4me3, H3K9me3, and H3K27me3 on tissue microarrays containing paragangliomas/pheochromocytomas (n = 134) and hereditary and sporadic smooth muscle tumors (n = 56) in comparison to their normal counterparts. Our results demonstrate distinct loss of 5 hmC in tumor cells in SDH- and FH-deficient tumors. Loss of 5 hmC in SDH-deficient tumors was associated with nuclear exclusion of TET1, a known regulator of 5 hmC levels. Moreover, increased methylation of H3K9me3 occurred predominantly in the chief cell component of SDH mutant tumors, while no changes were seen in H3K4me3 and H3K27me3, data supported by in vitro knockdown of SDH genes. We also show for the first time that FH-deficient smooth muscle tumors exhibit increased H3K9me3 methylation compared to wildtype tumors. Our findings reveal broadly similar patterns of epigenetic deregulation in both FH- and SDH-deficient tumors, suggesting that defects in genes of the TCA cycle result in common mechanisms of inhibition of histone and DNA demethylases.

Metastatic pheochromocytoma continues to be an incurable disease, and treatment with conventional cytotoxic chemotherapy offers limited efficacy. In the present study, we evaluated a novel topoisomerase I inhibitor, LMP-400, as a potential treatment for this devastating disease. We found a high expression of topoisomerase I in human metastatic pheochromocytoma, providing a basis for the evaluation of a topoisomerase 1 inhibitor as a therapeutic strategy. LMP-400 inhibited the cell growth of established mouse pheochromocytoma cell lines and primary human tumor tissue cultures. In a study performed in athymic female mice, LMP-400 demonstrated a significant inhibitory effect on tumor growth with two drug administration regimens. Furthermore, low doses of LMP-400 decreased the protein levels of hypoxia-inducible factor 1 (HIF-1α), one of a family of factors studied as potential metastatic drivers in these tumors. The HIF-1α decrease resulted in changes in the mRNA levels of HIF-1 transcriptional targets. In vitro, LMP-400 showed an increase in the growth-inhibitory effects in combination with other chemotherapeutic drugs that are currently used for the treatment of pheochromocytoma. We conclude that LMP-400 has promising antitumor activity in preclinical models of metastatic pheochromocytoma and its use should be considered in future clinical trials.

In solid tumors, neoplastic cells grow in contact with the so-called tumor microenvironment. The interaction between tumor cells and the microenvironment causes reciprocal metabolic reprogramming and favorable conditions for tumor growth and metastatic spread. To obtain an experimental model resembling the in vivo conditions of the succinate dehydrogenase B subunit (SDHB)-mutated paragangliomas (PGLs), we evaluated the effects of SDHB silencing on metabolism and proliferation in the human neuroblastoma cell line (SK-N-AS), cultured alone or in association with human fibroblasts. Silencing caused a 70% decrease in protein expression, an almost complete loss of the complex specific enzymatic activity, and a significant increase in HIF1α and HIF2α expression; it thus resembled the in vivo tumor cell phenotype. When compared with WT SK-N-AS cells, SDHB-silenced cells showed an altered metabolism characterized by an unexpected significant decrease in glucose uptake and an increase in lactate uptake. Moreover, silenced cells exhibited a significant increase in cell proliferation and metalloproteinase activity. When co-cultured with human fibroblasts, control cells displayed a significant decrease in glucose uptake and a significant increase in cell proliferation as compared with their mono-cultured counterparts. These effects were even more evident in co-cultured silenced cells, with a 70% decrease in glucose uptake and a 92% increase in cell proliferation as compared to their mono-cultured counterparts. The present data indicate for the first time, to our knowledge, that SDHB impairment causes metabolic and functional derangement of neural-crest-derived tumor cells and that the microenvironment, here represented by fibroblasts, strongly affects their tumor metabolism and growth capacity., (© 2015 Society for Endocrinology.)

Mitochondrial dysfunction, due to mutations of the gene encoding succinate dehydrogenase (SDH), has been implicated in the development of adrenal phaeochromocytomas, sympathetic and parasympathetic paragangliomas, renal cell carcinomas, gastrointestinal stromal tumours and more recently pituitary tumours. Underlying mechanisms behind germline SDH subunit B (SDHB) mutations and their associated risk of disease are not clear. To investigate genotype-phenotype correlation of SDH subunit B (SDHB) variants, a homology model for human SDH was developed from a crystallographic structure. SDHB mutations were mapped, and biochemical effects of these mutations were predicted in silico. Results of structural modelling indicated that many mutations within SDHB are predicted to cause either failure of functional SDHB expression (p.Arg27*, p.Arg90*, c.88delC and c.311delAinsGG), or disruption of the electron path (p.Cys101Tyr, p.Pro197Arg and p.Arg242His). GFP-tagged WT SDHB and mutant SDHB constructs were transfected (HEK293) to determine biological outcomes of these mutants in vitro. According to in silico predictions, specific SDHB mutations resulted in impaired mitochondrial localisation and/or SDH enzymatic activity. These results indicated strong genotype-functional correlation for SDHB variants. This study reveals new insights into the effects of SDHB mutations and the power of structural modelling in predicting biological consequences. We predict that our functional assessment of SDHB mutations will serve to better define specific consequences for SDH activity as well as to provide a much needed assay to distinguish pathogenic mutations from benign variants., (© 2015 Society for Endocrinology.)

The majority of benign adrenal cortex lesions leading to Cushing syndrome are associated to one or another abnormality of the cAMP/cGMP-phosphodiesterase signaling pathway. Phosphodiesterases (PDEs) are key regulatory enzymes of intracellular cAMP/cGMP levels. These second messengers play important regulatory roles in controlling steroidogenesis in the adrenal. Disruption of PDEs has been associated with a number of adrenal diseases. Specifically, genetic mutations have been associated with benign adrenal lesions, leading to Cushing syndrome and/or related adrenal hyperplasias. A Genome Wide Association study, in 2006, led to the identification of mutations in 2 PDE genes: PDE8B and PDE11A; mutations in these 2 genes modulate steroidogenesis. Further human studies have identified PDE2 as also directly regulating steroidogenesis. PDE2 decreases aldosterone production. This review focuses on the most recent knowledge we have gained on PDEs and their association with adrenal steroidogenesis and altered function, through analysis of patient cohorts and what we have learned from mouse studies., (© Georg Thieme Verlag KG Stuttgart · New York.)

Pheochromocytomas and paragangliomas are rare neuroendocrine tumors which develop from chromaffin cells of the adrenal medulla and extra-adrenal sites, leading to excess catecholamine release and hypertension. Many of the tumors are characterized by a high vascularity, suggesting the possible implementation of anti-angiogenic therapies for patients. Here, the efficacy of the tyrosine kinase inhibitors sunitinib and sorafenib was investigated in vivo and in vitro. Oral treatment with either sunitinib or sorafenib (40mg/kg/day) for 14days induced a marked reduction in the volume and weight of PC12 pheochromocytoma cell tumor xenografts in mice. Assessment of tumoral neo-angiogenesis, assessed by morphometric analysis of the vascular network after CD31 immunolabeling, showed that both sunitinib and sorafenib reduced the microvessel area (-85% and -80%, respectively) and length (-80% and -78%, respectively) in treated compared to control tumors. In addition, the number of vessel nodes was significantly lower in treated tumors (-95% and -84%, respectively). Furthermore, cleaved caspase 3 immunolabeling revealed a marked increase in the number of apoptotic cells in tumors from treated animals. Sunitinib and sorafenib could exert a direct effect on PC12 cell viability in vitro. While sunitinib induced a rapid (4h) and pronounced (5-fold) increase in caspase-3/7-dependent apoptosis, sorafenib seems to exert its cytotoxic activity through a different mechanism. Altogether, our data demonstrate that sunitinib and sorafenib have the ability to impair pheochromocytoma development by inhibiting angiogenesis and reducing tumor cell viability. These results strongly suggest that both sunitinib and sorafenib could represent valuable therapeutic tools for pheochromocytoma., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Context: Mutations of succinate dehydrogenase A/B/C/D genes (SDHx) increase susceptibility to development of pheochromocytomas and paragangliomas (PPGLs), with particularly high rates of malignancy associated with SDHB mutations., Objective: We assessed whether altered succinate dehydrogenase product-precursor relationships, manifested by differences in tumor ratios of succinate to fumarate or other metabolites, might aid in identifying and stratifying patients with SDHx mutations., Design, Setting, and Patients: PPGL tumor specimens from 233 patients, including 45 with SDHx mutations, were provided from eight tertiary referral centers for mass spectrometric analyses of Krebs cycle metabolites., Main Outcome Measure: Diagnostic performance of the succinate:fumarate ratio for identification of pathogenic SDHx mutations., Results: SDH-deficient PPGLs were characterized by 25-fold higher succinate and 80% lower fumarate, cis-aconitate, and isocitrate tissue levels than PPGLs without SDHx mutations. Receiver-operating characteristic curves for use of ratios of succinate to fumarate or to cis-aconitate and isocitrate to identify SDHx mutations indicated areas under curves of 0.94 to 0.96; an optimal cut-off of 97.7 for the succinate:fumarate ratio provided a diagnostic sensitivity of 93% at a specificity of 97% to identify SDHX-mutated PPGLs. Succinate:fumarate ratios were higher in both SDHB-mutated and metastatic tumors than in those due to SDHD/C mutations or without metastases., Conclusions: Mass spectrometric-based measurements of ratios of succinate:fumarate and other metabolites in PPGLs offer a useful method to identify patients for testing of SDHx mutations, with additional utility to quantitatively assess functionality of mutations and metabolic factors responsible for malignant risk.

The chemopreventive and antineoplastic activities of caffeic acid derivatives are highly dependent on the chemical structures and cancer cell types. The objective of the present study was to investigate the cytotoxicity of bornyl caffeate and the underlying molecular mechanisms in rat pheochromocytoma PC12 cells. Our initial studies demonstrated that bornyl caffeate exhibited potent cytotoxicity in PC12 cells in a concentration- and time-dependent manner. By examining the cell morphology on a fluorescence microscope and detecting the cell surface phosphoserine with Annexin V-FITC, we proposed that bornyl caffeate could induce apoptosis in PC12 cells. We tested this hypothesis by investigating the effects of bornyl caffeate on several apoptosis-related biomarkers. These experiments showed that bornyl caffeate induced the up-regulation of Bax and down-regulation of Bcl-xl, the disruption of mitochondrial membrane potential, the activation of caspase 3 and the cleavage of PARP. Mechanistic studies further revealed that bornyl caffeate caused the depletion of glutathione (GSH), generation of superoxide ion and progressive activation of p38 mitogen-activate protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in a concentration-dependent manner. In particular, GSH depletion appeared to be the most important mechanism underlying the cytotoxicity of bornyl caffeate. The preservation of the intracellular GSH contents with N-acetyl-L-cysteine (NAC), GSH and vitamin C abolished the effect of bornyl caffeate on the activation of p38 MAPK and JNK, preserved the integrity of mitochondrial membrane and ultimately rescued the cells from drug-induced cell death. These results suggest that bornyl caffeate induces apoptosis in PC12 cells via stimulating the depletion of GSH, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial transmembrane potential., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Pheochromocytomas and paragangliomas are neuroendocrine tumors shown to be responsive to multitargeted tyrosine kinase inhibitor (TKI) treatment. Despite growing knowledge regarding their genetic basis, the ability to predict behavior in these tumors remains challenging. There is also limited knowledge of their tyrosine kinase receptor expression and whether the clinical response observed to the TKI sunitinib relates only to its anti-angiogenic properties or also due to a direct effect on tumor cells. To answer these questions, an in vitro model of sunitinib treatment of a pheochromocytoma cell line was created. Sunitinib targets (VEGFRs, PDGFRs, and C-KIT), FGFRs, and cell cycle regulatory proteins were investigated in human tissue microarrays. SDHB immunohistochemistry was used as a surrogate marker for the presence of succinate dehydrogenase mutations. The FGFR4 G388R single nucleotide polymorphism was also investigated. Sunitinib treatment in vitro decreases cell proliferation mainly by targeting cell cycle, DNA metabolism, and cell organization genes. FGFR1, -2, and -4, VEGFR2, PDGFRα, and p16 were overexpressed in primary human pheochromocytomas and paragangliomas. Discordant results were observed for VEGFR1, p27, and p21 overexpressed in paragangliomas but underexpressed in pheochromocytomas; PDGFRβ, Rb, and Cyclin D1 overexpressed in paragangliomas only; and FGFR3 overexpressed in pheochromocytomas and underexpressed in paragangliomas. Low expression of C-KIT, p53, and Aurora kinase A and B was observed. Nuclear FGFR2 expression was associated with increased risk of metastasis (odds ratio (OR)=7.61, P=0.008), as was membranous PDGFRα (OR=13.71, P=0.015), membranous VEGFR1 (OR=8.01, P=0.037), nuclear MIB1 (OR=1.26, P=0.008), and cytoplasmic p27 (OR=1.037, P=0.030). FGFR3, VEGFR2, and C-KIT levels were associated with decreased risk of metastasis. We provide new insights into the mechanistic actions of sunitinib in pheochromocytomas and paragangliomas, and support current evidence that multitargeted TKIs might be a suitable treatment alternative for these tumors.

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Corticotropin-independent Cushing's syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood., Methods: We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro., Results: Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595&#95;596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing's syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased., Conclusions: Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).

Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia are the two characteristic types of primary aldosteronism. Dysregulation of adrenal cortical cell proliferation contributes to both diseases. We previously demonstrated that APA expressed less dopamine D2 receptor than the respective non-tumor tissue and might contribute to the overproduction of aldosterone. As activation of D2 receptor inhibits the proliferation of various cells, downregulation of D2 receptor in APA may play a role in the tumorigenesis of APA. In this study, we demonstrate that D2 receptor plays a role in angiotensin II (AII)-stimulated adrenal cortical cell proliferation. The D2 receptor agonist, bromocriptine, inhibited AII-stimulated cell proliferation in primary cultures of the normal human adrenal cortex and APA through attenuating AII-induced phosphorylation of PK-stimulated cyclin D1 protein expression and cell proliferation. D2 receptor also inhibited AII-induced ERK1/2 phosphorylation. Our results demonstrate that, in addition to inhibiting aldosterone synthesis/production, D2 receptor exerts an anti-proliferative effect in adrenal cortical and APA cells by attenuating PKCμ and ERK phosphorylation. The lower level of expression of D2 receptor in APA may augment cell proliferation and plays a crucial role in the tumorigenesis of APA. Our novel finding suggests a new therapeutic target for primary aldosteronism.

Purpose: This study aims to evaluate the role of F-DOPA PET/CT in staging and follow-up of paraganglioma syndromes succinate dehydrogenase (SDH)-mutation-related patients, comparing F-DOPA PET/CT results with morphological imaging and biochemical results., Patients and Methods: We retrospectively studied 10 consecutive patients (3 F, 7 M, mean age 32 yrs), all with a genetically demonstrated SDH mutation (5 SDH-D, 4 SDH-B, and 1 SDH-C) and all addressed to F-DOPA PET/CT scan. Seven patients had already been operated on for one or more pheochromocytomas and/or paragangliomas and were submitted to F-DOPA PET/CT scan according to clinical, biochemical, or radiological suspicion of recurrence, while 3 were only genetically positive, with no previous symptom/sign of the disease. For all patients, biochemical analysis (plasma and/or urinary catecholamine) and results of high-resolution morphological imaging studies (CT and/or MRI) were available. Histologic/cytologic findings or imaging and biochemical follow-up were taken as gold standard in all cases., Results: Seven out of 10 patients showed one or more areas of pathological F-DOPA accumulation. PET/CT demonstrated the presence of the disease in 4/6 patients with no increase in catecholamine levels ("biochemically silent"). Positive detection rate was 100% in SDH-D and 40% in "non-SDHD". Analyzing per lesion, F-DOPA PET/CT demonstrated more lesions than anatomical imaging (16 vs. 7) especially in head and neck paragangliomas., Conclusions: F-DOPA PET/CT seems to be the more accurate method for staging and restaging patients with SDH-mutations-related paraganglioma syndromes. F-DOPA is particularly useful in detecting head and neck and biochemically silent paragangliomas, and also in apparently healthy mutation-carrying people.

Methamphetamine (METH) is one of the most commonly abused agents by illicit-drug users. Thioredoxin-1 (Trx-1) plays important biological roles both in intra- and extracellular compartments, including in regulation of various intracellular molecules via thiol redox control. In this study, we found that Trx-1 was induced by METH in rat pheochromocytoma PC12 cells. Furthermore, PI3K/Akt pathway was involved in METH-induced increase of Trx-1 expression. An increase in phosphorylated cAMP response element-binding protein (CREB) was also observed after exposure of PC12 cells to METH, which was inhibited by a PI3K inhibitor, LY294002. In addition, the siRNA targeted toTrx-1 reduced the level of phosphorylated CREB by METH, suggesting Trx-1 is necessary for increased activity of CREB by METH. The results obtained in this study showed that Trx-1 might play a role in the actions of METH.

Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-L-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.

We analyzed the expression profiles of several steroidogenic enzymes in normal adrenals, aldosterone-producing adenomas (APA), cortisol-producing adenomas combined with Cushing's syndrome (CPA) or with subclinical Cushing's syndrome (SCPA), and nonfunctioning adrenal adenomas (NFA) to clarify the nature and characteristics of steroidogenesis in APA. Clinical data were collected for all subjects. In resected adrenal glands (normal adrenals, APA, CPA, SCPA, and NFA), the mRNA expression levels of the CYP17, HSD3B2, CYP11B1, and CYP11B2 genes were studied using real-time quantitative PCR and immunohistochemistry. The CYP11B2 mRNA level in APA was significantly higher than that in other groups. The CYP17/HSD3B2 ratio for mRNA in APA was significantly lower than those in the other groups. Low ratio of CYP17/HSD3B2 with high expression of CYP11B2 seems to explain steroidogenic characteristics of APA.

The present study aimed to investigate the expression of aldosterone synthase (CYP11B2), adrenocorticotropic hormone receptor (ACTH-R) and their regulating transcription factors in adrenal incidentalomas (AIs) from normotensive and hypertensive patients to distinguish subclinical or atypical primary aldosteronism (PA) from AIs. Total RNA was extracted from 8 normal adrenal cortices (NAs), 46 AIs, 15 aldosterone-producing adenomas (APAs) and 6 idiopathic hyperaldosteronisms (IHAs). Real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry were performed to determine the mRNA and protein expression of CYP11B2, ACTH-R, steroidogenic factor-1 (SF1) and dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) in the different tissues. The AI hypertensive subgroup displayed increased plasma aldosterone concentration (PAC) and PAC/PRA ratio (ARR) and decreased plasma renin activity (PRA) compared to the normotensive group. CYP11B2, ACTH-R and SF1 mRNAs were significantly higher in the APA group compared to the other groups, and gradually increased in AI hypertensive samples. DAX-1 mRNA was expressed faintly in PA compared with NA. In normotensive-AI samples, DAX-1 mRNA was higher compared to PA and AI hypertensive samples. Significant differences in gene expression levels in AIs were observed between probable and improbable PA patients. Immunohistochemical results were consistent with those of real-time PCR. Plasma aldosterone levels were positively correlated with CYP11B2, ACTH-R and SF1 mRNA and inversely correlated with DAX-1 mRNA. In conclusion, a significant number of hypertensive-AI patients may have subclinical forms of PA. CYP11B2, ACTH-R and their regulating transcription factors may be noteworthy in distinguishing subclinical PA from AIs.

Phaeochromocytomas (PCCs) are benign neuroendocrine tumours of the adrenal medulla. Approximately 10% of PCC patients develop metastases, but this frequency is much higher in specific subtypes of patients. The reliable diagnosis of malignant PCC can only be made after identification of a metastasis. To study the effect of Trp53 inactivation on PCC pathogenesis in Pten KO mice, we investigated the adrenals of a large cohort of mice with conditional monoallelic and biallelic inactivation of Trp53 and Pten. The adrenal weights were determined for all mice, and in a proportion of these mice, immunohistochemistry for tyrosine hydroxylase and dopamine β-hydroxylase was performed on the adrenals and corresponding lungs. Finally, comparative genomic hybridization (CGH) was performed. The histological and immunohistochemical results confirmed that the adrenal tumours were PCCs. Inactivation of one or both alleles of Trp53 resulted in earlier tumour occurrence in the Pten(loxP/loxP) mice as well as in the Pten(loxP/+) mice. In addition, lung metastases were found in up to 67% of mice. The CGH results showed that the most frequent genomic alterations were loss of chromosome 19 (86%) and gain of chromosome 15 (71%). In this study, we have shown that Pten/Trp53 KO mice showed metastatic PCC at high frequency and primary tumours occurred at younger ages in mice with Trp53 inactivation. Therefore, the present model appears to be a suitable model that might allow the preclinical study of new therapeutics for these tumours.

Systemic analysis of somatic mutations of other susceptibility genes in syndromic tumors as well as apparently sporadic tumors in well-characterized specimens is lacking. Its clinical relevance has not been studied. Our objective was to determine the frequency of second allele inactivation in syndromic tumors and determine the frequency and potential clinical impact of somatic mutations and loss of heterozygosity (LOH) of the known susceptibility genes in syndromic and sporadic tumors. Nine tumor specimens from clinically characterized VHL mutation, five from SDHB mutation, four from SDHD mutation, two from RET mutation carriers, and eight from apparently sporadic cases were analyzed. Tumor DNA mutation screening of the SDHx, VHL, and RET genes and LOH analyses of the SDHx and VHL genes were performed. The Yates-corrected chi-squared test was used for comparison of the clinical data and the molecular-genetic results. Second allele inactivation in tumors was identified in 83% of VHL, 80% of SDHB, and 50% of SDHD specimen. High prevalence of VHL (6/6, p=0.024) and SDHB (7/7, p=0.018) somatic mutations has been identified in the sporadic group compared to all others. In the group of the VHL tumors the SDHB somatic events were significantly lower (2/6; p=0.045). In 18/19 (95%) of cases, we were able to demonstrate the presence of at least two concomitant affected susceptibility genes. We conclude that LOH is the most prevalent second allele-inactivating event. SDHB and VHL somatic mutation might play a role in the sporadic forms of tumor development. There is no clinical impact of mutation screening or LOH analysis of tumor specimens.

Multiple endocrine neoplasia type 2a results from an activating germline mutation in the RET proto-oncogene. Carriers of a RET mutation are at risk of medullary thyroid carcinoma, pheochromocytoma, and primary hyperparathyroidism. Most individuals with multiple endocrine neoplasia type 2a eventually develop medullary thyroid carcinoma and as there is a strong genotype-phenotype correlation, guidelines have been established as to the age recommended for prophylactic thyroidectomy. However for rare mutations in the RET proto-oncogene there is insufficient evidence to provide guidance as to the risk of medullary thyroid carcinoma. We present a family with the rare RET mutation, D631Y in which the proband initially presented with a pheochromocytoma, and review the available literature pertaining to this mutation. In 83% of index cases, pheochromocytoma was the presenting feature and only 37% of adult germline mutation carriers have developed medullary thyroid carcinoma, none of whom have been reported to have nodal or metastatic disease. Patients with a D631Y RET mutation typically present with pheochromocytoma and medullary thyroid carcinoma appears to occur with a later onset than reported with other RET mutations. Based on the current literature we recommend performing prophylactic total thyroidectomy by age 12 years for D631Y carriers although this recommendation may need to be reviewed as additional data becomes available., (© Georg Thieme Verlag KG Stuttgart · New York.)

Currently, the diagnosis of malignant pheochromocytoma can only be made when there is clinical evidence of metastasis or extensive local invasion. Thus, there is a need for new diagnostic marker(s) to identify tumors with malignant potential. The purpose of this study was to identify microRNAs (miRNAs) that are differentially expressed between benign and malignant pheochromocytomas and assess their diagnostic accuracy. Toward this aim, we analyzed miRNA expression in benign and malignant pheochromocytoma tumor samples using whole genome microarray profiling. Microarray analysis identified eight miRNAs that were significantly differentially expressed between benign and malignant pheochromocytomas. We measured a subset of these miRNAs directly by RT-PCR and found that miR-483-5p, miR-183, and miR-101 had significantly higher expression in malignant tumors as compared to their benign counterparts. Area under the receiver operating curve (AUC) analysis indicated that miR-483-5p, miR-101, and miR-183 could be useful diagnostic markers for distinguishing malignant from benign pheochromocytomas. In addition, these miRNAs could be detected in pheochromocytoma patient serum. Overall our data suggest that misexpression of miR-483-5p, miR-101, and miR-183 is associated with malignant pheochromocytoma.

Aims: High expression of carbonic anhydrase IX (CAIX) is reported for clear cell renal cell carcinoma (RCC), with a paucity of data for non-renal genitourinary or adrenal tumours. This study investigated the immunohistochemical expression of CAIX throughout the genitourinary tract and adrenal gland., Methods and Results: High expression in the renal cortex was restricted to clear cell, papillary and clear cell papillary RCC and carcinoid. Core biopsies of clear cell RCC were consistently positive. Positivity within the urothelial tract was seen in urothelial carcinoma including squamous, small-cell, sarcomatoid and adenomatous differentiation and clear cell adenocarcinoma. Signet ring and plasmacytoid variants of urothelial carcinoma were negative. Phaeochromocytoma, adrenal cortical adenoma, seminoma, yolk sac tumour, choriocarcinoma, Leydig cell tumour and prostatic adenocarcinoma were predominately negative, with variable reactivity in adrenal cortical carcinoma, embryonal carcinoma, teratoma and Sertoli cell tumour., Conclusions: Carbonic anhydrase IX is a sensitive marker for clear cell RCC in core biopsies. However, other genitourinary or adrenal tumours that can have a clear cell appearance including urothelial, squamous cell, clear cell adeno and adrenal cortical carcinoma and Sertoli cell tumour express CAIX. Knowledge of expression overlap between these entities may prevent incorrect interpretation of immunohistochemical results, particularly if limited tissue is available., (© 2011 Blackwell Publishing Limited.)

Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children. MYCN amplification and increased BDNF/TrkB signaling are features of high-risk tumors; yet, only ˜25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. As aminoacylase 1 (ACY1), an amino acid deacetylase, is a putative tumor suppressor in small cell lung and renal cell carcinomas, we investigated whether it or the other family members aspartoacylase (ASPA, aminoacylase 2) or aminoacylase 3 (ACY3) could serve a similar function in neuroblastoma. Aminoacylase expression was examined in TrkB-positive, MYCN-amplified (SMS-KCNR and SK-N-BE) and TrkB-negative, non-MYCN-amplified (SK-N-AS, SK-N-SH, SH-SY5Y and SH-EP) neuroblastoma cell lines. Each aminoacylase exhibited distinct spatial localization (i.e., cytosolic ACY1, membrane-associated ASPA and nuclear ACY3). When SK-N-SH cells were treated with neural differentiation agents (e.g., retinoic acid and cAMP) in media containing 10% serum, ACY1 was the only aminoacylase whose expression was upregulated. ASPA was primarily expressed in SH-EP cells of a glial sublineage. ACY3 was more highly expressed in the TrkB-positive, MYCN-amplified lines. All three aminoacylases were expressed in normal human adrenal gland, a common site of neuroblastoma origin, but only ACY1 and ACY3 displayed detectable expression in primary neuroblastoma tumor. Bioinformatics data mining of Kaplan-Meier survival revealed that high ACY3 expression is correlated with poor prognosis, whereas low expression of ACY1 or ASPA is correlated with poor prognosis. These data suggest that aminoacylase expression is dysregulated in neuroblastoma., (Copyright © 2010 UICC.)

Background: We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation., Methods: Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma., Results: Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood., Conclusions: Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.

Receptor tyrosine kinase (RTK) inhibitors have revolutionized the treatment of metastatic renal cell carcinoma (mRCC) and significantly extended survival in these patients. Sunitinib is an oral multitargeted inhibitor of vascular endothelial growth factor receptors (VEGFRs-1, -2, and -3), platelet-derived growth factor receptors (PDGFRs-α and -β), stem-cell factor receptor (KIT), FMS-like tyrosine kinase 3 (FLT3), colony-stimulating factor 1 receptor (CSF-1R), and glial cell line-derived neurotrophic factor receptor (REarranged during Transfection; RET). Sunitinib is approved multinationally for the treatment of advanced RCC, and is considered the reference standard of care for first-line treatment. In clinical trials, sunitinib has been associated with a consistent, distinct profile of adverse events. Here we describe three cases that show that it is possible to manage adverse events occurring during sunitinib therapy, and thus allow patients with mRCC to receive an effective dose of sunitinib in order to achieve long-term disease control. These cases also show that surgical resection, performed whenever possible, can help to improve control of metastatic disease and so avoid the unnecessary toxicity and high costs of prolonged antiangiogenic therapy.

Background: The failure of cytotoxic cancer regimens to cure the most drug-resistant, well-differentiated solid tumors has been attributed to the heterogeneity of cell types that differ in their capacities for growth, differentiation, and metastases. We investigated the effect of LB1, a small molecule inhibitor of serine/threonine protein phosphatase 2A (PP2A), on its ability to inhibit a low growth fraction and highly drug-resistant solid neuroendocrine tumor, such as metastatic pheochromocytoma (PHEO). Subsequently, we evaluated the increased efficacy of chemotherapy combined with LB1., Methodology/principal Findings: The effect of LB1 and temozolomide (TMZ), a standard chemotherapeutic agent that alone only transiently suppressed the growth and regression of metastatic PHEO, was evaluated in vitro on a single PHEO cell line and in vivo on mouse model of metastatic PHEO. In the present study, we show that metastatic PHEO, for which there is currently no cure, can be eliminated by combining LB1, thereby inhibiting PP2A, with TMZ. This new treatment approach resulted in long term, disease-free survival of up to 40% of animals bearing multiple intrahepatic metastases, a disease state that the majority of patients die from. Inhibition of PP2A was associated with prevention of G1/S phase arrest by p53 and of mitotic arrest mediated by polo-like kinase 1 (Plk-1)., Conclusions/significance: The elimination of DNA damage-induced defense mechanisms, through transient pharmacologic inhibition of PP2A, is proposed as a new approach for enhancing the efficacy of non-specific cancer chemotherapy regimens against a broad spectrum of low growth fraction tumors very commonly resistant to cytotoxic drugs.

Primary aldosteronism (PA), an autonomous aldosterone hypersecretion from adrenal adenoma and/or hyperplasia, and subclinical Cushing syndrome (SCS), a mild but autonomous cortisol hypersecretion from adrenal adenoma without signs or symptoms of Cuhing's syndrome, are now well-recognized clinical entities of adrenal incidentaloma. However, the clinicopathological features of PA associated with SCS (PA/SCS) remain unknown. The present study was undertaken to study the prevalence of PA/SCS among PA patients diagnosed at our institute, and characterize their clinicopathlogical features. The prevalence of PA/SCS was 8 of 38 PA patients (21%) studied. These 8 PA/SCS patients were significantly older and had larger tumor, higher serum potassium levels, lower basal plasma levels of aldosterone, ACTH and DHEA-S as well as lower response of aldosterone after ACTH stimulation than those in 12 patients with aldosterone-producing adenoma without hypercortisolism. All 8 PA/SCS patients showed unilateral uptake by adrenal scintigraphy at the ipsilateral side, whereas the laterality of aldosterone hypersecretion as determined by adrenal venous sampling varied from ipsilateral (3), contralateral (2), and bilateral side (2). 6 PA/SCS patinets who underwent adrenalectomy required hydrocortisone replacement postoperatively. Histopathological analysis of the resected adrenal tumors from 5 PA/SCS patients revealed a single adenoma in 3, and double adenomas in 2, with varying degrees of positive immunoreactivities for steroidgenic enzymes (3β-HSD, P450(C17)) by immunohistochemical study as well as CYP11B2 mRNA expression as measured by real-time RT-PCR. In conclusion, PA/SCS consists of a variety of adrenal pathologies so that therapeutic approach differs depending on the disease subtype., (©The Japan Endocrine Society)

Expression of carboxypeptidase E (CPE), a prohormone processing enzyme in different cancer types, was analyzed from data in the GEO profile database (http://www.ncbi.nlm.nih.gov/geo/) and experimentally in pheochromocytomas. Analysis of microarray data demonstrated that significantly elevated levels of CPE mRNA was found in many metastatic non-endocrine cancers: cervical, colon rectal, renal cancers, Ewing sarcomas (bone cancer), and various types of astrocytomas and oligodendrogliomas, whereas expression of CPE mRNA was virtually absent in their respective counterpart normal tissues. Moreover, there was higher CPE mRNA expression in cells from the metastatic tumor compared to those from the primary tumor in colorectal cancer. Elevated CPE mRNA expression was found in neuroendocrine tumors in lung and pituitary adenomas, although the significance is unclear since endocrine and neuroendocrine cells normally express CPE. However, studies of neuroendocrine tumors, pheochromocytomas, revealed expression of not only wild-type CPE, but a variant which was correlated with tumor behavior. Extremely high CPE mRNA copy numbers of the variant were found in very large or invasive tumors, both of which usually indicate poor prognosis. Thus, collectively the data suggest that CPE may play a role in promoting tumor growth and invasion. CPE could potentially serve as a diagnostic and prognostic biomarker for metastasis in different cancer types.

Prediction of malignant behaviour of pheochromocytomas/sympathetic paragangliomas (PCCs/PGLs) is very difficult if not impossible on a histopathological basis. In a familial setting, it is well known that succinate dehydrogenase subunit B (SDHB)-associated PCC/PGL very often metastasise. Recently, absence of SDHB expression as measured through immunohistochemistry was shown to be an excellent indicator of the presence of an SDH germline mutation in PCC/PGL. SDHB loss is believed to lead to tumour formation by activation of hypoxia signals. To clarify the potential use of SDHB immunohistochemistry as a marker of malignancy in PCC/PGL and its association with classic hypoxia signalling we examined SDHB, hypoxia inducible factor-1α (Hif-1α) and its targets CA-9 and GLUT-1 expression on protein level using immunohistochemistry on a tissue micro array on a series of familial and sporadic tumours of 115 patients. Survival data was available for 66 patients. SDHB protein expression was lost in the tumour tissue of 12 of 99 patients. Of those 12 patients, 5 had an SDHB germline mutation, in 4 patients no germline mutation was detected and mutational status remained unknown in parts in 3 patients. Loss of SDHB expression was not associated with increased classic hypoxia signalling as detected by Hif-1α, CA-9 or GLUT-1 staining. Loss of SDHB expression was associated with an adverse outcome. The lack of correlation of SDHB loss with classic hypoxia signals argues against the current hypoxia hypothesis in malignant PCC/PGL. We suggest SDHB protein loss as a marker of adverse outcome both in sporadic and in familial PCC/PGL.