Human Pheochromocytoma Research Articles

Near-Infrared Emissive Super Penetrating Conjugated Polymer Dots for Intratumoral Imaging in 3D Tumor Spheroid Models.

This study describes the formation of single-chain polymer dots (Pdots) via ultrasonic emulsification of nonionic donor-acceptor-donor type (D-A-D) alkoxy thiophene-benzobisthiadiazole-based conjugated polymers (Poly BT) with amphiphilic cetyltrimethylammonium bromide (CTAB). The methodology yields Pdots with a high cationic surface charge (+56.5mV ± 9.5) and average hydrodynamic radius of 12nm. Optical characterization reveals that these Pdots emit near-infrared (NIR) light at a maximum wavelength of 860nm owing to their conjugated polymer backbone consisting of D-A-D monomers. Both colloidal and optical properties of these Pdots make them promising fluorescence emissive probes for bioimaging applications. The significant advantage of positively charged Pdots is demonstrated in diffusion-limited mediums such as tissues, utilizing human epithelial breast adenocarcinoma, ATCC HTB-22 (MCF-7), human bone marrow neuroblastoma, ATCC CRL-2266 (SH-SY5Y), and rat adrenal gland pheochromocytoma, CRL-1721 (PC-12)tumor spheroid models. Fluorescence microscopy analysis of tumor spheroids from MCF-7, SH-SY5Y, and PC-12 cell lines reveals the intensity profile of Pdots, confirming extensive penetration into the central regions of the models. Moreover, a comparison with mitochondria staining dye reveals an overlap between the regions stained by Pdots and the dye in all three tumor spheroid models. These results suggest that single-chain D-A-D type Pdots, cationized via CTAB, exhibit long-range mean free path of penetration (≈1µm) in dense mediums and tumors.

The Role of Adrenomedullin as a Predictive Marker of the Risk of Death and Adverse Clinical Events: A Review of the Literature.

Adrenomedullin (ADM) is a vasodilatory peptide that plays a crucial role in maintaining cardiovascular health through its various biological functions. ADM was discovered in the acidic extract of human pheochromocytoma tissue and has been recognized for its significant effects on the vascular system. The main functions of ADM include vasodilation, controlling blood pressure and maintaining vascular integrity, although its role on cardiovascular health is broader. Research has shown that elevated levels of adrenomedullin have been observed in a large number of severe diseases, with high risk of death. In this work, we examined the role of ADM as a predictive molecule of the risk of mortality and adverse clinical outcome through a narrative review of the scientific literature. The results were divided based on the pathologies and anatomical districts examined. This review demonstrates how ADM shows, in many diseases and different systems, a close correlation with the risk of mortality. These results prove the value of ADM as a prognostic marker in various clinical contexts and diseases, with utility in the stratification of the risk of clinical worsening and/or death and in the evaluation of therapeutic efficacy. The results open new perspectives with respect to the concrete possibility that ADM enters clinical practice as an effective diagnostic and prognostic marker of death as well as a molecular target for therapies aimed at patient survival.

Synthesis of Novel Plant-Derived Encapsulated Radiolabeled Compounds for the Diagnosis of Parkinson's Disease and the Evaluation of Biological Effects with In Vitro/In Vivo Methods.

Parkinson's disease (PD) is a neurodegenerative disorder that affects millions of individuals globally. It is characterized by the loss of dopaminergic neurons in Substantia Nigra pars compacta (SNc) and striatum. Neuroimaging techniques such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) help diagnosing PD. In this study, the focus was on developing technetium-99m ([99mTc]Tc) radiolabeled drug delivery systems using plant-derived compounds for the diagnosis of PD. Madecassoside (MA), a plant-derived compound, was conjugated with Levodopa (L-DOPA) to form MA-L-DOPA, which was then encapsulated using Poly Lactic-co-Glycolic Acid (PLGA) to create MA-PLGA and MA-L-DOPA-PLGA nanocapsules. Extensive structural analysis was performed using various methods such as Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), dynamic light scattering (DLS), and scanning electron microscopy (SEM) to characterize the synthesized products. Radiochemical yields of radiolabeled compounds were determined using thin layer radio chromatography (TLRC) and high performance liquid radio chromatography (HPLRC) methods. In vitro cell culture studies were conducted on human neuroblastoma (SH-SY5Y) and rat pheochromocytoma (PC-12) cell lines to assess the incorporation of [99mTc]Tc radiolabeled compounds ([99mTc]Tc-MA, [99mTc]Tc-MA-L-DOPA, [99mTc]Tc-MA-PLGA and [99mTc]Tc-MA-L-DOPA-PLGA) and the cytotoxicity of inactive compounds (MA and MA-L-DOPA compounds and encapsulated compounds (MA-PLGA and MA-L-DOPA-PLGA). Additionally, the biodistribution studies were carried out on healthy male Sprague-Dawley rats and a Parkinson's disease experimental model to evaluate the compounds' bioactivity using the radiolabeled compounds. The radiochemical yields of all radiolabeled compounds except [99mTc]Tc-L-DOPA-PLGA were above 95% and had stability over 6h. The cytotoxic effects of all substances on SH-SY5Y and PC-12 cells increase with increasing concentration values. The uptake values of PLGA-encapsulated compounds are statistically significant in SH-SY5Y and PC-12 cells. The biodistribution studies showed that [99mTc]Tc-MA is predominantly retained in specific organs and brain regions, with notable uptake in the prostate, muscle, and midbrain. PLGA-encapsulation led to higher uptake in certain organs, suggesting its biodegradable nature may enhance tissue retention, and surface modifications might further optimize brain penetration. Overall, the results indicate that radiolabeled plant-derived encapsulated drug delivery systems with [99mTc]Tc hold potential as diagnostic agents for PD symptoms. This study contributes to the advancement of drug delivery agents in the field of brain research.

Identification of potential molecular targets for the treatment of cluster 1 human pheochromocytoma and paraganglioma via comprehensive proteomic characterization

BackgroundPheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. New drug targets and proteins that would assist sensitive PPGL imagining could improve therapy and quality of life of patients with PPGL, namely those with recurrent or metastatic disease. Using a combined proteomic strategy, we looked for such clinically relevant targets among integral membrane proteins (IMPs) upregulated on the surface of tumor cells and non-membrane druggable enzymes in PPGL.MethodsWe conducted a detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor lysate, which provides information largely on non-membrane proteins, was accompanied by specific membrane proteome-aimed methods, namely glycopeptide enrichment using lectin-affinity, glycopeptide capture by hydrazide chemistry, and enrichment of membrane-embedded hydrophobic transmembrane segments.ResultsThe study identified 67 cell surface integral membrane proteins strongly upregulated in PPGL compared to control chromaffin tissue. We prioritized the proteins based on their already documented direct role in cancer cell growth or progression. Increased expression of the seven most promising drug targets (CD146, CD171, ANO1, CD39, ATP8A1, ACE and SLC7A1) were confirmed using specific antibodies. Our experimental strategy also provided expression data for soluble proteins. Among the druggable non-membrane enzymes upregulated in PPGL, we identified three potential drug targets (SHMT2, ARG2 and autotaxin) and verified their upregulated expression.ConclusionsApplication of a combined proteomic strategy recently presented as “Pitchfork” enabled quantitative analysis of both, membrane and non-membrane proteome, and resulted in identification of 10 potential drug targets in human PPGL. Seven membrane proteins localized on the cell surface and three non-membrane druggable enzymes proteins were identified and verified as significantly upregulated in PPGL. All the proteins have been previously shown to be upregulated in several human cancers, and play direct role in cancer progression. Marked upregulation of these proteins along with their localization and established direct roles in tumor progression make these molecules promising candidates as drug targets or proteins for sensitive PPGL imaging.

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas.

The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4-8 week period but the persistence of small numbers of synaptophysin/tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin-positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS.

Background: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis. Methods: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology. Results: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs. Conclusions: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases.

Somatostatin analogue pasireotide (SOM230) inhibits catecholamine secretion in human pheochromocytoma cells.

Increasingly common, neuroendocrine tumors (NETs) are regarded nowadays as neoplasms potentially causing debilitating symptoms and life-threatening medical conditions. Pheochromocytoma is a NET that develops from chromaffin cells of the adrenal medulla, and is responsible for an excessive secretion of catecholamines. Consequently, patients have an increased risk for clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Somatostatin analogues are among the main anti-secretory medical drugs used in current clinical practice in patients with NETs. However, their impact on pheochromocytoma-associated catecholamine hypersecretion remains incompletely explored. This study investigated the potential efficacy of octreotide and pasireotide (SOM230) on human tumor cells directly cultured from freshly resected pheochromocytomas using an implemented catecholamine secretion measurement by carbon fiber amperometry. SOM230 treatment efficiently inhibited nicotine-induced catecholamine secretion both in bovine chromaffin cells and in human tumor cells whereas octreotide had no effect. Moreover, SOM230 specifically decreased the number of exocytic events by impairing the stimulation-evoked calcium influx as well as the nicotinic receptor-activated inward current in human pheochromocytoma cells. Altogether, our findings indicate that SOM230 acts as an inhibitor of catecholamine secretion through a mechanism involving the nicotinic receptor and might be considered as a potential anti-secretory treatment for patients with pheochromocytoma.

Antiproliferative effects of metformin in cellular models of pheochromocytoma

Pheochromocytomas (PCCs) are rare neuroendocrine tumors derived from adrenal medulla chromaffin cells. Malignancy and recurrence are rare but demand effective treatment. Metformin exerts antiproliferative effects in several cancer cell lines. We thus evaluated the effects of metformin on cell viability and proliferation, cellular respiration and AMPK-AKT-mTOR-HIFA proliferation pathway on a rat PCC cell line (PC12-Adh). We then addressed metformin's effects on the AMPK-AKT-mTOR-HIFA pathway on two human primary cultures: one from a VHL-mutant PCC and other from a sporadic PCC. Metformin (20 mM) inhibited PC12-Adh cell proliferation, and decreased oxygen consumption, ATP production and proton leak, in addition to loss of mitochondrial membrane potential. Further, metformin induced AMPK phosphorylation and impaired AMPK-PI3k-AKT-mTOR pathway activation. The mTOR pathway was also inhibited in human VHL-related PCC cells, however, in an AMPK-independent manner. Metformin-induced decrease of HIF1A levels was likely mediated by proteasomal degradation. Altogether our results suggest that metformin impairs PCC cellular proliferation.

Germline SUCLG2 Variants in Patients With Pheochromocytoma and Paraganglioma.

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. 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. 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. 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.

Novel Germline SUCLG2 Mutations in Patients With Pheochromocytoma and Paraganglioma

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors derived from neural crest cells that are frequently linked to mutations including those in Krebs cycle enzymes, particularly succinate dehydrogenase (SDH). Succinyl-CoA ligase (SUCL) catalyzes reversible conversion of succinyl-CoA to succinate providing the substrate for SDH. While mitochondrial diseases were documented for the mutations in SUCL subunits G1 and A2, the association of GDP/GTP-specific subunit SUCLG2 mutations with specific pathologies including cancer have not been reported. In our study, 352 patients with apparently sporadic PPGLs underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health. Additionally, human pheochromocytoma (hPheo1) cells were used for gene manipulation to produce SUCLG2 knock-out (KO). Tumor tissues and hPheo1 SUCLG2 KO cells were used for further analysis focusing on mechanism of germline variants effect on mitochondrial functions. We detected eight germline SUCLG2 mutations in 15 patients which represents 4.3% of the cohort. Germline variants together with LOH led to decreased levels of SDH subunit B resulting in aberrant respiration and accumulation of succinate, well recognized oncometabolite. Manipulation of SUCLG2 in hPheo1 cells confirmed decrease in SDHB leading to faulty assembly of mitochondrial complex II and alteration of its respiration and activity. In summary, our study identified an association between SUCLG2 and PPGL. Larger scale sequencing and uncovering additional cases bearing SUCLG2 variants will further clarify the relationship between SUCLG2 and SDHx, particularly SDHB, as well as their role in disease etiology.

Succinate Mediates Tumorigenic Effects via Succinate Receptor 1: Potential for New Targeted Treatment Strategies in Succinate Dehydrogenase Deficient Paragangliomas.

Paragangliomas and pheochromocytomas (PPGLs) are chromaffin tumors associated with severe catecholamine-induced morbidities. Surgical removal is often curative. However, complete resection may not be an option for patients with succinate dehydrogenase subunit A-D (SDHx) mutations. SDHx mutations are associated with a high risk for multiple recurrent, and metastatic PPGLs. Treatment options in these cases are limited and prognosis is dismal once metastases are present. Identification of new therapeutic targets and candidate drugs is thus urgently needed. Previously, we showed elevated expression of succinate receptor 1 (SUCNR1) in SDHB PPGLs and SDHD head and neck paragangliomas. Its ligand succinate has been reported to accumulate due to SDHx mutations. We thus hypothesize that autocrine stimulation of SUCNR1 plays a role in the pathogenesis of SDHx mutation-derived PPGLs. We confirmed elevated SUCNR1 expression in SDHx PPGLs and after SDHB knockout in progenitor cells derived from a human pheochromocytoma (hPheo1). Succinate significantly increased viability of SUCNR1-transfected PC12 and ERK pathway signaling compared to control cells. Candidate SUCNR1 inhibitors successfully reversed proliferative effects of succinate. Our data reveal an unrecognized oncometabolic function of succinate in SDHx PPGLs, providing a growth advantage via SUCNR1.

Activation of RAS Signalling is Associated with Altered Cell Adhesion in Phaeochromocytoma.

Phaeochromocytomas and paragangliomas (PPGLs) are neuroendocrine catecholamine-producing tumours that may progress into inoperable metastatic disease. Treatment options for metastatic disease are limited, indicating a need for functional studies to identify pharmacologically targetable pathophysiological mechanisms, which require biologically relevant experimental models. Recently, a human progenitor phaeochromocytoma cell line named “hPheo1” was established, but its genotype has not been characterised. Performing exome sequencing analysis, we identified a KIF1B T827I mutation, and the oncogenic NRAS Q61K mutation. While KIF1B mutations are recurring somatic events in PPGLs, NRAS mutations have hitherto not been detected in PPGLs. Therefore, we aimed to assess its implications for the hPheo1 cell line, and possible relevance for the pathophysiology of PPGLs. We found that transient downregulation of NRAS in hPheo1 led to elevated expression of genes associated with cell adhesion, and enhanced adhesion to hPheo1 cells’ extracellular matrix. Analyses of previously published mRNA data from two independent PPGL patient cohorts (212 tissue samples) revealed a subcluster of PPGLs featuring hyperactivated RAS pathway-signalling and under-expression of cell adhesion-related gene expression programs. Thus, we conclude that NRAS activity in hPheo1 decreases adhesion to their own extracellular matrix and mirrors a transcriptomic RAS-signalling-related phenomenon in PPGLs.

SDHB and SDHA Immunohistochemistry in Canine Pheochromocytomas

Simple SummaryPheochromocytomas are adrenal tumors that occur in both dogs and people. One of the more common gene families involved in the development of this tumor in people is succinate dehydrogenase (SDH). In people, immunohistochemistry can be used with biopsy samples to predict gene pathways that may be involved in the development of the tumor. This is faster and cheaper than performing extensive sequencing to determine if genes are involved. We tested 35 dog tumors to determine how likely SDH mutations were. While our data suggest significant numbers of SDH mutations, these mutations do not appear to be associated with tumor aggression.Pheochromocytomas (PCs) are tumors arising from the chromaffin cells of the adrenal glands and are the most common tumors of the adrenal medulla in animals. In people, these are highly correlated to inherited gene mutations in the succinate dehydrogenase (SDH) pathway; however, to date, little work has been done on the genetic basis of these tumors in animals. In humans, immunohistochemistry has proven valuable as a screening technique for SDH mutations. Human PCs that lack succinate dehydrogenase B (SDHB) immunoreactivity have a high rate of mutation in the SDH family of genes, while human PCs lacking succinate dehydrogenase A (SDHA) immunoreactivity have mutations in the SDHA gene. To determine if these results are similar for dogs, we performed SDHA and SDHB immunohistochemistry on 35 canine formalin-fixed, paraffin-embedded (FFPE) PCs. Interestingly, there was a loss of immunoreactivity for both SDHA and SDHB in four samples (11%), suggesting a mutation in SDHx including SDHA. An additional 25 (71%) lacked immunoreactivity for SDHB, while retaining SDHA immunoreactivity. These data suggest that 29 out of the 35 (82%) may have an SDH family mutation other than SDHA. Further work is needed to determine if canine SDH immunohistochemistry on PCs correlates to genetic mutations that are similar to human PCs.

Understanding the Molecular Underpinnings of Neuronal Kinase CDK5 mediated Metabolic Vulnerabilities in Neuroendocrine Tumors

BackgroundAberrantly activated neuronal kinase CDK5 invoke alternations in mitochondrial dynamics and metabolic dysfunction associated with neurological disorders. Although pro‐tumorigenic role of CDK5 has been recently defined but there is insufficient data to understand the mechanistic role of CDK5 in cancer associated mitochondrial structure and function.HypothesisIn this study we intend to investigate putative signalling hotspots downstream of aberrantly activated CDK5 in neuroendocrine tumors of adrenal glands called Pheochromocytoma (PC).ApproachOur preliminary data for the first time showed deregulation of Ca2+ homeostasis and overexpression of CDK5 and its co‐activators‐p35/25 in malignant PC tumors inflicted with loss‐of‐function mutations in mitochondrial enzyme‐Succinate dehydrogenase B (SDHB). To elucidate role of CDK5 in tumorigenesis, we created a novel bi‐transgenic mouse model where doxycycline inducible overexpression of CDK5/p25 (p25OE) developed organ specific PC lesions.ResultsPhopshoproteomic analysis on p25OE PC tumors unraveled a tumor suppressive mechanism modulated by novel phosphorylation site on metabolic protein‐PRKAG2 (S‐65). PRKAG2 is a non‐catalytic regulatory subunit of AMP‐activated protein kinase (AMPK). S‐65 site on PRKAG2 were found downregulated in human PC tumors compared to normal adrenal medulla. Further biochemical characterization uncovered CDK5‐GSK3β‐AMPK crosstalk underlying human PC cellular proliferation. Picking CDK5 pocket with a novel CDK5 inhibitor ‘MRT3‐007’ induced anti‐proliferative effects with simultaneous increase in phosphorylation states of PRKAG2(S‐65) / AMPK(T‐172) sites followed by excessive mitochondrial fission.ConclusionThese intriguing initial findings set the ground to discover therapeutic vulnerabilities associated with CDK5/p25/PRKAG2/AMPK signaling cascade and Mitochondrial Dynamics in Neuroendocrine tumors.Schematic depiction of working model demonstrating a novel crosstalk between a metabolic sensor ‘PRKAG2/AMPK’ and ‘Mitochondrial Fission’ synapsed via neuronal kinase ‘CDK5/p25’ in Pheochromocytoma (PC tumors) inflicted with mutations in SDHB gene.Figure 1

Expression of the ret Proto-oncogene in Human Medullary Thyroid Carcinomas and Pheochromocytomas of MEN 2A

We studied the expression of the ret proto-oncogene (proto-ret) in human medullary thyroid carcinomas (MTCs) and pheochromocytomas of multiple endocrine neoplasia type 2A (MEN 2A) by Northern blot analysis. Expression of the normal-sized transcripts was detected in all 12 MTCs and in 6 of 8 pheochromocytomas. In situ localization of proto-ret mRNA revealed that the signal was confined to the cytoplasm of MTC cells. By Southern blot analysis neither amplification nor gross genetic changes of proto-ret were found in the tumors. Although no transcripts were detected in the normal portion of the thyroid from one MEN 2A patient, faint signals were detected in normal adrenal glands by Northern blot analysis, probably due to minor populations of C-cells and chromaffin cells in specimens from which MTC and pheochromocytoma might later develop. Proto-ret may play an important role in differentiation of a specific cell lineage from neuroectoderm, and it may be involved in development of MEN 2A tumors.

Evaluation of the effect of herbal extracts and their bioactive compounds against motion sickness by regulating neurotransmitter levels in vitro and in vivo

Motion sickness (MS) is caused due to sensory mismatch. Sensory conflict theory which proves acetylcholine, histamine elevations as the primary reason for MS provides a path for an effective pharmaco-therapy, by an attempt to inhibit the elevation of acetylcholine, histamine during travel. The present study aimed to screen herbal extracts and their bioactive compounds for anti-MS effect by using in vitro and in vivo models. Ten herbal extracts and their bioactive compounds were tested for its effect on acetylcholine esterase (AChE) activity, histamine and dopamine release in human leukaemia KU812 and rat pheochromocytoma PC12 cells respectively. Rutin hydrate, menthol, eugenol, linalool and hesperidin showed less inhibitory effect on AChE activity and promoting the degradation of acetylcholine and inhibited the release of histamine and dopamine from KU812 and PC12 cells. These bioactive compounds being present in Ocimum basilicum L. var. basilicum seed extract (OBSE) was chosen for further validation studies in vivo. Anti-MS effect was evaluated in BALB/c mice model using conditioned taste aversion. Pre-treatment with OBSE at 300 mg/kg bodyweight concentration prevented MS by reducing acetylcholine, histamine, dopamine, epinephrine levels, cholinergic receptor muscarinic 1 gene expression and increasing serotonin and norepinephrine levels and thus it could serve as a potent herbal antidote for MS.

Abstract LB-C04: Phosphoproteomics unravels dynamic regulation of energy sensor by CDK5 in Neuroendocrine Tumor

Abstract Background: Pheochromocytoma (PC) is a rare Neuroendocrine (NE) tumor usually originates from adrenal gland’s chromaffin cells. These tumors are often benign but can become malignant causing excessive secretion of catecholamines leading to life threatening situation. Pro-tumorigenic role of CDK5, a proline-directed serine/threonine neuronal kinase has been implicated in tumor development. However, the broader downstream signalling events contributing to the function of CDK5 are largely unknown in NE tumors, particularly Pheochromocytoma. Results-Here we showed for the first time that aberrant CDK5/p25 signaling attributes human PC lesions both sporadic and in tumours’ incorporating mutations in key susceptibility genes such as SDHx and VHL. Unique doxycycline regulated bi-transgenic (TG) mouse model system was designed to drive CDK5/p25 overexpression (OE) in a NE cell specific manner localized within adrenal medulla. PC lesions developed in the TG mouse model accurately recapitulate human PC pathobiology and thus rendered a platform for discovering precision use of novel CDK5 inhibitors. Picking CDK5 pocket with a novel CDK5 inhibitor ‘MRT3-007’ induced significant tumor regression in TG/ SDHB knockdown xenograft/ & metastatic PC models. Next, to identify putative CDK5 substrates, we analysed phosphoproteomic profile of [p25‘turned On’/ Proliferating] versus [p25‘turned Off’/ Arrested] PC lesions. The analyses identified dynamic downregulation of 122 phosphosites in proliferating tumors. Further interrogation of biological relevance of top 5 downregulated phosphorylation sites unleashed anti-proliferative function of Phospho-Ser-65 (pS65) on PRKAG2 which is a non-catalytic regulatory gamma subunit of metabolic regulator- AMP-activated protein kinase (AMPK). Biochemical characterization uncovered mysterious CDK5-GSK3β-AMPK crosstalk regulating proliferation of human PC (hPheo1) cells. Preliminary findings show that our hit compound inhibits CDK5 with simultaneous elevation of novel phosphorylation on pS65-PRKAG2 embarking catalytic activation of AMPK kinase. Moreover, in vitro kinase data suggests that CDK5 influences activation of AMPK enzymatic components via regulation of pS9/21-GSK3β activation dephosphorylation. Once activated, AMPK invokes anti-proliferative response by promoting excessive mitochondrial fission in PC cells. In Conclusion, this study illuminates comprehensive view of phosphorylation events downstream of complex CDK5 signaling in human PC. Identification of novel CDK5/p25-GSK3-PRKAG2-AMPK signalling cascade may provide new insights into target-directed Neuroendocrine Cancer Therapeutics. Citation Format: PRIYANKA GUPTA, KEEHN STRANGE, RAHUL TELENGE, LAURENT MEIJER, HANS GHAYEE, KAREL PACAK, SUSHANTH REDDY, JAMES BIBB. Phosphoproteomics unravels dynamic regulation of energy sensor by CDK5 in Neuroendocrine Tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C04. doi:10.1158/1535-7163.TARG-19-LB-C04

Pheochromocytoma as a Clinical Model of Peripheral Sympathetic Overdrive: Old and New Findings.

The present paper will review the results of experimental and clinical studies aimed at defining the functional behavior of the central and peripheral nervous system in adrenal pheochromocytoma. The contribution of sympathetic neural influences to the development of high blood pressure values in pheochromocytoma is complex. Studies performed in experimental animal models have shown that hypertension and the concomitant high circulating levels of catecholamines can lead to inhibition of central sympathetic neural outflow by reflex mechanisms and direct stimulation of central adrenergic receptors, respectively. However, these studies have also shown that high circulating levels of catecholamines favor a downregulation of alpha- and beta-adrenergic receptors, lessening their response to endogenous and exogenous adrenergic stimulation. The present paper reviews results of human studies performed by our group and others on the behavior of the central and peripheral nervous system in human pheochromocytoma. We discuss data collected in patients with different levels of peripheral sympathetic drive, i.e., before and after surgical removal of the adrenal pheochromocytoma. In the presence of elevated plasma catecholamine level, such as that characterizing adrenal pheochromocytoma, microneurography shows that central sympathetic neural activity is normal or even inhibited. At the peripheral vascular level, pheochromocytoma is characterized by a reduced vascular reactivity to exogenous sympathetic stimulation but a normal response by the vessels to endogenous adrenergic stimulation.