Chemical composition, anti-inflammatory and cytotoxic activity of essential oils from two Luvunga species (L. scandens and L. hongiaoensis) from Vietnam.

New essential oils (EOs) extracted from different parts of two Luvunga species (L. scandens and L. hongiaoensis) from Vietnam were investigated for their chemical composition, anti-inflammatory and cytotoxic activity. Sixty-nine total compounds were identified in the EOs by GC/MS. The major constituent of the leaf, fruit, and root EOs from L. scandens was ß-caryophyllene (71.5%, 63.0%, and 31.5% respectively). The main compounds in L. hongiaoensis EOs were ß-elemene (34.3% in leaf oil) and caryophyllene oxide (21.2% in root oil, 19.4% in stem oil). The EO from L. scandens fruits significantly inhibited nitric oxide production on LPS-induced RAW264.7 cells (IC50 = 37.95 ± 2.76 µg/mL). The EOs from L. hongiaoensis roots and L. scandens leaves and fruits exhibited cytotoxic activity against MCF-7, SK-LU-1, and HepG2 (IC50 from 49.74 ± 3.36 to 97.82 ± 8.61 µg/mL). This is the first report on L. hongiaoensis EOs and significantly complements the composition and bioactivity of L. scandens EOs.

Immune signature of pheochromocytoma and paraganglioma in context of neuroendocrine neoplasms associated with prognosis.

PURPOSE: To understand prognostic immune cell infiltration signatures in neuroendocrine neoplasms (NENs), particularly pheochromocytoma and paraganglioma (PCPG), we analyzed tumor transcriptomic data from The Cancer Genome Atlas (TCGA) and other published tumor transcriptomic data of NENs. METHODS: We used CIBERSORT to infer immune cell infiltrations from bulk tumor transcriptomic data from PCPGs, in comparison to gastroenteropancreatic neuroendocrine tumors (GEPNETs) and small cell lung carcinomas (SCLCs). PCPG immune signature was validated with NanoString immune panel in an independent cohort. Unsupervised clustering of the immune infiltration scores from CIBERSORT was used to find immune clusters. A prognostic immune score model for PCPGs and the other NENs were calculated as a linear combination of the estimated infiltration of activated CD8+/CD4+ T cells, activated NK cells, and M0 and M2 macrophages. RESULTS: In PCPGs, we found five dominant immune clusters, associated with M2 macrophages, monocytes, activated NK cells, M0 macrophages and regulatory T cells, and CD8+/CD4+ T cells respectively. Non-metastatic tumors were associated with activated NK cells and metastatic tumors were associated with M0 macrophages and regulatory T cells. In GEPNETs and SCLCs, M0 macrophages and regulatory T cells were associated with unfavorable outcomes and features, such as metastasis and high-grade tumors. The prognostic immune score model for PCPGs and the NENs could predict non-aggressive and non-metastatic diseases. In PCPGs, the immune score was also an independent predictor of metastasis-free survival in a multivariate Cox regression analysis. CONCLUSION: The transcriptomic immune signature in PCPG correlates with clinical features like metastasis and prognosis.

A long noncoding RNA-microRNA expression signature predicts metastatic signature in pheochromocytomas and paragangliomas.

PURPOSE: In hopes of discovering new markers for metastatic or aggressive phenotypes of pheochromocytomas and paragangliomas (PCPG), we analyzed the noncoding transcriptome from patient gene expression data in The Cancer Genome Atlas. METHODS: Differential expression of miRNAs was observed between PCPG molecular subtypes. We specifically characterized candidate miRNAs that are upregulated in pseudohypoxic PCPGs with mutations in succinate dehydrogenase complex subunits, B and/or D (SDHB and/or SDHD, respectively), which are mutations associated with unfavorable clinical outcomes. RESULTS: Our computational analysis identified four candidate miRNAs that showed elevated expression in metastatic compared to non-metastatic PCPGs: miR-182, miR-183, miR-96, and miR-383. We also found six candidate lncRNAs harboring opposite expression patterns from the miRNAs when we analyzed the expression profiles of their predicted target lncRNAs. Three of these lncRNA candidates, USP3-AS1, LINC00877, and AC009312.1, were validated to have reduced expression in metastatic compared to non-metastatic PCPGs. Finally, using univariate and multivariate analysis, we found miRNA miR-182 to be an independent predictor of metastasis-free survival in PCPGs. CONCLUSIONS: We identified candidate miRNA and lncRNAs associated with metastasis-free survival in PCPGs.

Germline SUCLG2 Variants in Patients With Pheochromocytoma and Paraganglioma.

BACKGROUND: Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II [CII]), a known tumor suppressor in PPGL. METHODS: A cohort of 352 patients with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2. RESULTS: We describe 8 germline variants in the guanosine triphosphate-binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of SDH, and faulty assembly of the complex II, resulting in aberrant respiration and elevated succinate accumulation. CONCLUSIONS: Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance.

Identification of Immune Cell Infiltration in Murine Pheochromocytoma during Combined Mannan-BAM, TLR Ligand, and Anti-CD40 Antibody-Based Immunotherapy.

Immunotherapy has become an essential component in cancer treatment. However, the majority of solid metastatic cancers, such as pheochromocytoma, are resistant to this approach. Therefore, understanding immune cell composition in primary and distant metastatic tumors is important for therapeutic intervention and diagnostics. Combined mannan-BAM, TLR ligand, and anti-CD40 antibody-based intratumoral immunotherapy (MBTA therapy) previously resulted in the complete eradication of murine subcutaneous pheochromocytoma and demonstrated a systemic antitumor immune response in a metastatic model. Here, we further evaluated this systemic effect using a bilateral pheochromocytoma model, performing MBTA therapy through injection into the primary tumor and using distant (non-injected) tumors to monitor size changes and detailed immune cell infiltration. MBTA therapy suppressed the growth of not only injected but also distal tumors and prolonged MBTA-treated mice survival. Our flow cytometry analysis showed that MBTA therapy led to increased recruitment of innate and adaptive immune cells in both tumors and the spleen. Moreover, adoptive CD4+ T cell transfer from successfully MBTA-treated mice (i.e., subcutaneous pheochromocytoma) demonstrates the importance of these cells in long-term immunological memory. In summary, this study unravels further details on the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination.

Therapeutic Targeting of SDHB-Mutated Pheochromocytoma/Paraganglioma with Pharmacologic Ascorbic Acid.

PURPOSE: Pheochromocytomas and paragangliomas (PCPG) are usually benign neuroendocrine tumors. However, PCPGs with mutations in the succinate dehydrogenase B subunit (SDHB) have a poor prognosis and frequently develop metastatic lesions. SDHB-mutated PCPGs exhibit dysregulation in oxygen metabolic pathways, including pseudohypoxia and formation of reactive oxygen species, suggesting that targeting the redox balance pathway could be a potential therapeutic approach. EXPERIMENTAL DESIGN: We studied the genetic alterations of cluster I PCPGs compared with cluster II PCPGs, which usually present as benign tumors. By targeting the signature molecular pathway, we investigated the therapeutic effect of ascorbic acid on PCPGs using in vitro and in vivo models. RESULTS: By investigating PCPG cells with low SDHB levels, we show that pseudohypoxia resulted in elevated expression of iron transport proteins, including transferrin (TF), transferrin receptor 2 (TFR2), and the divalent metal transporter 1 (SLC11A2; DMT1), leading to iron accumulation. This iron overload contributed to elevated oxidative stress. Ascorbic acid at pharmacologic concentrations disrupted redox homeostasis, inducing DNA oxidative damage and cell apoptosis in PCPG cells with low SDHB levels. Moreover, through a preclinical animal model with PCPG allografts, we demonstrated that pharmacologic ascorbic acid suppressed SDHB-low metastatic lesions and prolonged overall survival. CONCLUSIONS: The data here demonstrate that targeting redox homeostasis as a cancer vulnerability with pharmacologic ascorbic acid is a promising therapeutic strategy for SDHB-mutated PCPGs.

Targeting NRF2-Governed Glutathione Synthesis for SDHB-Mutated Pheochromocytoma and Paraganglioma.

Succinate dehydrogenase subunit B (SDHB) deficiency frequently occurs in cluster I pheochromocytomas and paragangliomas (PCPGs). SDHB-mutated PCPGs are characterized by alterations in the electron transport chain, metabolic reprogramming of the tricarboxylic cycle, and elevated levels of reactive oxygen species (ROS). We discovered that SDHB-deficient PCPG cells exhibit increased oxidative stress burden, which leads to elevated demands for glutathione metabolism. Mechanistically, nuclear factor erythroid 2-related factor 2 (NRF2)-guided glutathione de novo synthesis plays a key role in supporting cellular survival and the proliferation of SDHB-knockdown (SDHBKD) cells. NRF2 blockade not only disrupted ROS homeostasis in SDHB-deficient cells but also caused severe cytotoxicity by the accumulation of DNA oxidative damage. Brusatol, a potent NRF2 inhibitor, showed a promising effect in suppressing SDHBKD metastatic lesions in vivo, with prolonged overall survival in mice bearing PCPG allografts. Our findings highlight a novel therapeutic strategy of targeting the NRF2-driven glutathione metabolic pathway against SDHB-mutated PCPG.

Long intergenic noncoding RNA profiles of pheochromocytoma and paraganglioma: A novel prognostic biomarker.

Many long intergenic noncoding RNAs (lincRNAs) serve as cancer biomarkers for diagnosis or prognostication. To understand the role of lincRNAs in the rare neuroendocrine tumors pheochromocytoma and paraganglioma (PCPG), we performed first time in-depth characterization of lincRNA expression profiles and correlated findings to clinical outcomes of the disease. RNA-Seq data from patients with PCPGs and 17 other tumor types from The Cancer Genome Atlas and other published sources were obtained. Differential expression analysis and a machine-learning model were used to identify transcripts specific to PCPGs, as well as established PCPG molecular subtypes. Similarly, lincRNAs specific to aggressive PCPGs were identified, and univariate and multivariate analysis was performed for metastasis-free survival. The results were validated in independent samples using RT-PCR. From a pan-cancer context, PCPGs had a specific and unique lincRNA profile. Among PCPGs, five different molecular subtypes were identified corresponding to the established molecular classification. Upregulation of 13 lincRNAs was found to be associated with aggressive/metastatic PCPGs. RT-PCR validation confirmed the overexpression of four lincRNAs in metastatic compared to non-metastatic PCPGs. Kaplan-Meier analysis identified five lincRNAs as prognostic markers for metastasis-free survival of patients in three subtypes of PCPGs. Stratification of PCPG patients with a risk-score formulated using multivariate analysis of lincRNA expression profiles, presence of key driver mutations, tumor location, and hormone secretion profiles showed significant differences in metastasis-free survival. PCPGs thus exhibit a specific lincRNA expression profile that also corresponds to the established molecular subgroups and can be potential marker for the aggressive/metastatic PCPGs.

The Significant Reduction or Complete Eradication of Subcutaneous and Metastatic Lesions in a Pheochromocytoma Mouse Model after Immunotherapy Using Mannan-BAM, TLR Ligands, and Anti-CD40.

Therapeutic options for metastatic pheochromocytoma/paraganglioma (PHEO/PGL) are limited. Here, we tested an immunotherapeutic approach based on intratumoral injections of mannan-BAM with toll-like receptor ligands into subcutaneous PHEO in a mouse model. This therapy elicited a strong innate immunity-mediated antitumor response and resulted in a significantly lower PHEO volume compared to the phosphate buffered saline (PBS)-treated group and in a significant improvement in mice survival. The cytotoxic effect of neutrophils, as innate immune cells predominantly infiltrating treated tumors, was verified in vitro. Moreover, the combination of mannan-BAM and toll-like receptor ligands with agonistic anti-CD40 was associated with increased mice survival. Subsequent tumor re-challenge also supported adaptive immunity activation, reflected primarily by long-term tumor-specific memory. These results were further verified in metastatic PHEO, where the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 into subcutaneous tumors resulted in significantly less intense bioluminescence signals of liver metastatic lesions induced by tail vein injection compared to the PBS-treated group. Subsequent experiments focusing on the depletion of T cell subpopulations confirmed the crucial role of CD8+ T cells in inhibition of bioluminescence signal intensity of liver metastatic lesions. These data call for a new therapeutic approach in patients with metastatic PHEO/PGL using immunotherapy that initially activates innate immunity followed by an adaptive immune response.

Nonmosaic somatic HIF2A mutations associated with late onset polycythemia-paraganglioma syndrome: Newly recognized subclass of polycythemia-paraganglioma syndrome.

BACKGROUND: Somatic mutations in hypoxia-inducible factor 2α (HIF2A) are associated with polycythemia-paraganglioma syndrome. Specifically, the classic presentation of female patients with recurrent paragangliomas (PGLs), polycythemia (at birth or in early childhood), and duodenal somatostatinomas has been described. Studies have demonstrated that somatic HIF2A mutations occur as postzygotic events and some to be associated with somatic mosaicism affecting hematopoietic and other tissue precursors. This phenomenon could explain the development of early onset of polycythemia in the absence of erythropoietin-secreting tumors. METHODS: Correlation analysis was performed between mosaicism of HIF2A mutant patients and clinical presentations. RESULTS: Somatic HIF2A mutations (p.A530V, p.P531S, and p.D539N) were identified in DNA extracted from PGLs of 3 patients. No somatic mosaicism was detected through deep sequencing of blood genomic DNA. Compared with classic syndrome, both polycythemia and PGL in all 3 patients developed at an advanced age with polycythemia at age 30, 30, and 17 years and PGLs at age 34, 30, and 55 years, respectively. Somatostatinomas were not detected, and 2 patients had ophthalmic findings. The biochemical phenotype in all 3 patients was noradrenergic with 18 F-fluorodopa PET/CT as the most sensitive imaging modality. All patients demonstrated multiplicity, and none developed metastatic disease. CONCLUSION: These findings suggest that newer techniques need to be developed to detect somatic mosaicism in patients with this syndrome. Absence of HIF2A mosaicism in patients with somatic HIF2A mutations supports association with late onset of the disease, milder clinical phenotype, and an improved prognosis compared with patients who have HIF2A mosaicism.

Targeting NAD+/PARP DNA Repair Pathway as a Novel Therapeutic Approach to SDHB-Mutated Cluster I Pheochromocytoma and Paraganglioma.

Purpose: Cluster I pheochromocytomas and paragangliomas (PCPGs) tend to develop malignant transformation, tumor recurrence, and multiplicity. Transcriptomic profiling suggests that cluster I PCPGs and other related tumors exhibit distinctive changes in the tricarboxylic acid (TCA) cycle, the hypoxia signaling pathway, mitochondrial electron transport chain, and methylation status, suggesting that therapeutic regimen might be optimized by targeting these signature molecular pathways.Experimental Design: In the present study, we investigated the molecular signatures in clinical specimens from cluster I PCPGs in comparison with cluster II PCPGs that are related to kinase signaling and often present as benign tumors.Results: We found that cluster I PCPGs develop a dependency to mitochondrial complex I, evidenced by the upregulation of complex I components and enhanced NADH dehydrogenation. Alteration in mitochondrial function resulted in strengthened NAD+ metabolism, here considered as a key mechanism of chemoresistance, particularly, of succinate dehydrogenase subunit B (SDHB)-mutated cluster I PCPGs via the PARP1/BER DNA repair pathway. Combining a PARP inhibitor with temozolomide, a conventional chemotherapeutic agent, not only improved cytotoxicity but also reduced metastatic lesions, with prolonged overall survival of mice with SDHB knockdown PCPG allograft.Conclusions: In summary, our findings provide novel insights into an effective strategy for targeting cluster I PCPGs, especially those with SDHB mutations. Clin Cancer Res; 24(14); 3423-32. ©2018 AACR.

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2V617F positive polycythemia vera: a case report.

BACKGROUND: The role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome. CASE PRESENTATION: A female presented with a history of JAK2V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35-45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells. CONCLUSIONS: This is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.

Catecholamine metabolomic and secretory phenotypes in phaeochromocytoma.

Phaeochromocytomas and paragangliomas (PPGLs) are highly heterogeneous tumours with variable catecholamine biochemical phenotypes and diverse hereditary backgrounds. This analysis of 18 catecholamine-related plasma and urinary biomarkers in 365 patients with PPGLs and 846 subjects without PPGLs examined how catecholamine metabolomic profiles are impacted by hereditary background and relate to variable hormone secretion. Catecholamine secretion was assessed in a subgroup of 156 patients from whom tumour tissue was available for measurements of catecholamine contents. Among all analytes, the free catecholamine O-methylated metabolites measured in plasma showed the largest tumour-related increases relative to the reference group. Patients with tumours due to multiple endocrine neoplasia type 2 and neurofibromatosis type 1 (NF1) showed similar catecholamine metabolite and secretory profiles to patients with adrenaline-producing tumours and no evident hereditary background. Tumours from these three patient groups contained higher contents of catecholamines, but secreted the hormones at lower rates than tumours that did not contain appreciable adrenaline, the latter including PPGLs due to von Hippel-Lindau (VHL) and succinate dehydrogenase (SDH) gene mutations. Large increases of plasma dopamine and its metabolites additionally characterised patients with PPGLs due to the latter mutations, whereas patients with NF1 were characterised by large increases in plasma dihydroxyphenylglycol and dihydroxyphenylacetic acid, the deaminated metabolites of noradrenaline and dopamine. This analysis establishes the utility of comprehensive catecholamine metabolite profiling for characterising the distinct and highly diverse catecholamine metabolomic and secretory phenotypes among different groups of patients with PPGLs. The data further suggest developmental origins of PPGLs from different populations of chromaffin cell progenitors.

Low sensitivity of glucagon provocative testing for diagnosis of pheochromocytoma.

CONTEXT: Pheochromocytomas can usually be confirmed or excluded using currently available biochemical tests of catecholamine excess. Follow-up tests are, nevertheless, often required to distinguish false-positive from true-positive results. The glucagon stimulation test represents one such test; its diagnostic utility is, however, unclear. OBJECTIVE: The aim of the study was to determine the diagnostic power of the glucagon test to exclude or confirm pheochromocytoma. DESIGN, SETTING, AND SUBJECTS: Glucagon stimulation tests were carried out at three specialist referral centers in 64 patients with pheochromocytoma, 38 patients in whom the tumor was excluded, and in a reference group of 36 healthy volunteers. MAIN OUTCOME MEASURES: Plasma concentrations of norepinephrine and epinephrine were measured before and after glucagon administration. Several absolute and relative test criteria were used for calculating diagnostic sensitivity and specificity. Expression of the glucagon receptor was examined in pheochromocytoma tumor tissue from a subset of patients. RESULTS: Larger than 3-fold increases in plasma norepinephrine after glucagon strongly predicted the presence of a pheochromocytoma (100% specificity and positive predictive value). However, irrespective of the various criteria examined, glucagon-provoked increases in plasma catecholamines revealed the presence of the tumor in less than 50% of affected patients. Diagnostic sensitivity was particularly low in patients with pheochromocytomas due to von Hippel-Lindau syndrome. Tumors from these patients showed no significant expression of the glucagon receptor. CONCLUSION: The glucagon stimulation test offers insufficient diagnostic sensitivity for reliable exclusion or confirmation of pheochromocytoma. Because of this and the risk of hypertensive complications, the test should be abandoned in routine clinical practice.

Characterization of an animal model of aggressive metastatic pheochromocytoma linked to a specific gene signature.

Pheochromocytomas are chromaffin cell-derived neuroendocrine tumors. There is presently no cure for metastatic pheochromocytoma and no reliable way to distinguish malignant from benign tumors before the development of metastases. In order to successfully manage pheochromocytoma, it is necessary to better understand the biological determinants of tumor behavior. For this purpose, we have recently established a mouse model of metastatic pheochromocytoma using tail vein injection of mouse pheochromocytoma (MPC) cells. We optimized this model modifying the number of cells injected, length of trypsin pre-treatment, and incubation temperature and duration for the MPC cells before injection, and by serial passage and re-selection of tumors exhibiting the metastatic phenotype. We evaluated the effect of these modifications on tumor growth using serial in vivo Magnetic Resonance Imaging studies. These results show that number of cells injected, the pre-injection incubation temperature, and duration of trypsin treatment are important factors to produce faster growing, more aggressive tumors that yielded secondary metastatic lesions. Serial harvest, culture and re-selection of metastatic liver lesions produced even more aggressive pheochromocytoma cells that retained their biochemical phenotype. Microarray gene expression comparison and quantitative real-time PCR of these more aggressive cells to the MPC-parental cell line identified genes that may be important for the metastatic process.

Differential expression of the regulated catecholamine secretory pathway in different hereditary forms of pheochromocytoma.

Pheochromocytomas in patients with von Hippel-Lindau (VHL) syndrome and multiple endocrine neoplasia type 2 (MEN 2) differ in the types and amounts of catecholamines produced and the resulting signs and symptoms. We hypothesized the presence of different processes of catecholamine release reflecting differential expression of components of the regulated secretory pathway among the two types of hereditary tumors. Differences in catecholamine secretion from tumors in patients with VHL syndrome (n = 47) and MEN 2 (n = 32) were examined using measurements of catecholamines in tumor tissue, urine, and plasma, the last of which was under baseline conditions in all subjects and in a subgroup of patients who received intravenous glucagon to provoke catecholamine release. Microarray and proteomics analyses, quantitative PCR, and Western blotting were used to assess expression of tumor tissue secretory pathway components. The rate constant for baseline catecholamine secretion was 20-fold higher in VHL than in MEN 2 tumors (0.359 +/- 0.094 vs. 0.018 +/- 0.009 day(-1)), but catecholamine release was responsive only to glucagon in MEN 2 tumors. Compared with tumors from MEN 2 patients, those from VHL patients were characterized by reduced expression of numerous components of the regulated secretory pathway (e.g., SNAP25, syntaxin, rabphilin 3A, annexin A7, calcium-dependent secretion activator). The mutation-dependent differences in expression of secretory pathway components indicate a more mature regulated secretory pathway in MEN 2 than VHL tumors. These data provide a unique mechanistic link to explain how variations in the molecular machinery governing exocytosis may contribute to clinical differences in the secretion of neurotransmitters or hormones and the subsequent presentation of a disease.

Neuropeptide Y expression in phaeochromocytomas: relative absence in tumours from patients with von Hippel-Lindau syndrome.

Phaeochromocytomas are rare neuroendocrine tumours that produce catecholamines and numerous secretory proteins and peptides, including neuropeptide Y (NPY), a vasoactive peptide with influences on blood pressure. The production of catecholamines and NPY by phaeochromocytomas is highly variable. This study examined influences of hereditary factors and differences in catecholamine production on tumour expression of NPY, as assessed by quantitative PCR, enzyme immunoassay and immunohistochemistry. Phaeochromocytomas included hereditary adrenaline-producing tumours (adrenergic phenotype) in multiple endocrine neoplasia type 2 (MEN 2), predominantly noradrenaline-producing tumours (noradrenergic phenotype) in von Hippel-Lindau (VHL) syndrome, and other adrenergic and noradrenergic tumours where there was no clear hereditary syndrome. NPY levels in phaeochromocytomas from VHL patients were lower (P<0.0001) than in those from MEN 2 patients for both mRNA (84-fold difference) and the peptide (99-fold difference). These findings were supported by immunohistochemistry. NPY levels were also lower in VHL tumours than in those where there was no hereditary syndrome. Relative absence of expression of NPY in phaeochromocytomas from VHL patients when compared with other groups appears to be largely independent of differences in catecholamine production and is consistent with a unique phenotype in VHL syndrome.

Transcriptional regulation of phenylethanolamine N-methyltransferase in pheochromocytomas from patients with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2.

Pheochromocytomas in multiple endocrine neoplasia type 2 (MEN-2) express phenylethanolamine N-methyltransferase (PNMT), the enzyme that catalyzes conversion of norepinephrine to epinephrine, whereas those in von Hippel-Lindau (VHL) syndrome do not. Consequently, pheochromocytomas in MEN-2 produce epinephrine, whereas those in VHL syndrome produce mainly norepinephrine. This study examined whether transcription factors known to regulate expression of PNMT explain the different tumor phenotypes in these syndromes. Quantitative polymerase chain reaction (PCR) and Western blotting were used to assess levels of mRNA and protein for the glucocorticoid receptor, early growth response 1 (Egr-1), the Sp1 transcription factor (Sp1), and MYC-associated zinc finger protein (MAZ) in 6 MEN-2 and 13 VHL tumors. Results were cross-checked with data obtained using microarray gene expression profiling in a further set of 10 MEN-2 and 12 VHL tumors. Pheochromocytomas in MEN-2 and VHL syndrome did not differ in expression of the glucocorticoid receptor, Egr-1, Sp1, or MAZ as assessed by quantitative PCR and Western blotting. Microarray data also indicated no relevant differences in expression of the glucocorticoid receptor, Egr-1, MAZ, and the AP2 transcription factor. Thus, our results do not support a role for the above transcription factors in determining differences in expression of PNMT in pheochromocytomas from patients with VHL syndrome and MEN-2. Microarray analysis, however, did indicate differences in expression of genes involved in neural crest cell lineage and chromaffin cell development, consistent with differential survival of PNMT-expressing cells in the two syndromes.

Different expression of catecholamine transporters in phaeochromocytomas from patients with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2.

OBJECTIVE: Phaeochromocytomas in patients with multiple endocrine neoplasia type 2 (MEN 2) produce adrenaline, whereas those with von Hippel-Lindau (VHL) syndrome do not. This study assessed whether these distinctions relate to differences in expression of the transporters responsible for uptake and storage of catecholamines - the noradrenaline transporter and the vesicular monoamine transporters (VMAT 1 and VMAT 2). METHODS: Tumour tissue and plasma samples were obtained from 31 patients with hereditary phaeochromocytoma - 18 with VHL syndrome and 13 with MEN 2. We used quantitative PCR, Western blotting, electron microscopy, immunohistochemistry and measurements of plasma and tumour catecholamines to assess differences in expression of the transporters in noradrenaline-producing vs adrenaline-producing hereditary tumours. These differences were compared with those in a further group of 26 patients with non-syndromic phaeochromocytoma. RESULTS: Adrenaline-producing phaeochromocytomas in MEN 2 patients expressed more noradrenaline transporter mRNA and protein than noradrenaline-producing tumours in VHL patients. In contrast, there was greater expression of VMAT 1 in VHL than MEN 2 tumours, while expression of VMAT 2 did not differ significantly. These differences were associated with larger numbers of storage vesicles and higher tissue contents of catecholamines in MEN 2 than in VHL tumours. Differences in expression of the noradrenaline transporter were weaker, and those of VMAT 1 and VMAT 2 stronger, in noradrenaline and adrenaline-producing non-syndromic than in hereditary tumours. CONCLUSIONS: The findings show that, in addition to differences in catecholamine biosynthesis, phaeochromocytomas in MEN 2 and VHL syndrome also differ in expression of the transporters responsible for uptake and vesicular storage of catecholamines.