ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 63 GP6 | DOI: 10.1530/endoabs.63.GP6

Cellular modelling of SDH-associated pathomechanism of pheochromocytomas and paragangliomas

Balazs Sarkadi1,2, Katalin Meszaros2,3,4, Ildiko Krencz5, Sara Zakarias1, Kinga Nemeth1, Gabor Barja5, Anna Sebestyen5, Judit Papay5, Katalin Borka6, Zoltan Hujber5, Miklos Toth1, Peter Igaz1, Christos Chinopoulos7 & Attila Patocs2,3,4

12nd Department of Medicine, Semmelweis University, Budapest, Hungary; 2‘Lendület’ Hereditary Endocrine Tumours Research Group, HAS-SE, Budapest, Hungary; 3Department of Laboratory Medicine, Semmelweis University, Budapest, Hungary; 4Bionics Innovation Center, Budapest, Hungary; 51st Department of Pathology and Experimental Cancer, Semmelweis University, Budapest, Hungary; 62nd Department of Pathology, Semmelweis University, Budapest, Hungary; 7Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

Introduction: The capability of cancer to accommodate to special metabolic circumstances is a hallmark of it’s existence. Pheochromocytoma/paragangliomas (Pheo/PGL) are rare neuroendocrine tumors with strong and specific metabolic phenotype due to mutations of genes encoding succinate dehydrogenase (SDH) subunits. In this study our aim was to evaluate the expression of glutaminase-1 in hereditary Pheo/PGL tissues and to inhibit SDH activity via pharmacological treatments and gene silencing in order to identify an in vitro model useful for the evaluation of metabolic consequences of SDH inhibition.

Materials and methods: Immunohistochemistry was performed on 15 Pheo/PGL tumor tissue blocks obtained from 9 patients with hereditary Pheo/PGL. SDHB silencing was carried out using siRNA on PC12 cells. Itaconic acid (Ita) and atpenin A5 (AA5) were used as pharmacological inhibitors of SDH enzyme on PC12, HeLa and H295R cells. Metabolite profiles were assessed with liquid chromatography-mass spectrometry (LC-MS), data were normalized to DNA concentrations. Cellular viability was assessed with alamarBlue.

Results: The expression of glutaminase-1 (GLS-1) by immunohistochemistry positively correlated with malignancy in Pheo/PGL tumors. All SDH inhibiting treatments significantly increased the succinate/fumarate ratio in all cell lines compared to control. SDHB siRNA transfection significantly increased the cell viability of PC12 cells. Itaconate significantly increased the viability of PC12 cells but significantly decreased HeLa and H295R cells’ viability. Atpenin significantly decreased viability of H295R cells, and significantly increased HeLa cell line’s viability, while no effect in PC12 cells was observed. SDH inhibition had significant effects on the metabolite profiles, depending on the inhibition method and cell type. In PC12 cells glutamate levels showed significant decrease without lactate accumulation after Ita or SDHB siRNA treatments. siRNA inhibition resulted in decreased malate and aspartate levels. HeLa and H295R cells showed significantly higher lactate and glutamate and decreased aspartate concentrations upon Ita treatment, while malate concentrations only increased in HeLa cells. AA5 treatment didn’t yield cell specific results: all cell lines exhibited significantly increased lactate and decreased glutamate, malate and aspartate levels compared to controls.

Conclusion: Increased GLS-1 expression in malignant samples points to the importance of glutamine/glutamate metabolism in Pheo/PGL. Ita treatment of PC12 cells mimics the phenotype observed in SDHB mutated Pheo/PGL tissues. Dissecting SDH inhibition (Ita) from electron-transport chain blockage (AA5) caused cell type specific effects. Targeting glutamine metabolism might yield a novel therapeutic target for malignant Pheo/PGL.