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Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

ABSTRACT: Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chromaffin cell line), Hela (human cervix epithelial cell line), and H295R (human adrenocortical cell line) cells, we demonstrated that chromaffin cells were not affected negatively by the inhibition of SDH either by siRNA directed against SDHB or treatment with SDH inhibitors (itaconate and atpenin A5). Cell viability and intracellular metabolite measurements pointed to the cell line specific consequences of SDH impairment and to the importance of glutamate metabolism in chromaffin cells. A significant increase in glutaminase-1 (GLS-1) expression after SDH impairment was observed in PC12 cells. GLS-1 inhibitor BPTES was capable of significantly decreasing proliferation of SDH impaired PC12 cells. Glutaminase-1 and SDHB expressions were tested in 35 Pheo/PGL tumor tissues. Expression of GLS1 was higher in the SDHB low expressed group compared to SDHB high expressed tumors. Our data suggest that the SDH-associated malignant potential of Pheo/PGL is strongly dependent on GLS-1 expression and glutaminases may be novel targets for therapy.

SUBMITTER: Sarkadi B

PROVIDER: S-EPMC7139890 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

Sarkadi Balazs B Meszaros Katalin K Krencz Ildiko I Canu Letizia L Krokker Lilla L Zakarias Sara S Barna Gabor G Sebestyen Anna A Papay Judit J Hujber Zoltan Z Butz Henriett H Darvasi Otto O Igaz Peter P Doczi Judit J Luconi Michaela M Chinopoulos Christos C Patocs Attila A

Cancers 20200305 3

Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the <i>SDHB</i> subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chroma ...[more]

PMID: 32150977

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Project description:Introduction: Genotype constitutes a pivotal factor in the comprehensive management of pheochromocytomas and paragangliomas (PPGLs). This underlines the need to (1) further elucidate the genetic etiology of these pathologies, (2) investigate the underlying pathogenic mechanisms, and (3) identify therapeutic targets. These goals rely on generating and studying relevant biological models, particularly in vivo. However, this has remained a disappointment using traditional animal models. In this context, there is a need to develop fast, reliable in vivo models of PPGLs which display clinically relevant biochemical hallmarks of PPGLs. Objective: Our study aims to establish zebrafish larvae as a relevant and convenient proxy for the functional characterization of PPGL-associated genetic variants. Methods: We took advantage of the genetic accessibility of zebrafish embryos to generate first-generation (F0) loss-of-function models for sdhb, a canonical PPGL-associated gene, using CRISPR/CAS9 technology. F0 zebrafish embryos were microinjected with a CRISPR/CAS9 molecular cocktail containing sdhb-specific guide RNAs and compared with control embryos injected with non-active CRISPR/CAS9. The mutagenic score of our CRISPR/CAS9 approach was validated by High-Resolution Melting analysis, and mutant zebrafish larvae were phenotyped during their first five days of development. Notably, the levels of catecholamines/metanephrines (in whole fish extract and bathing medium) and Krebs cycle metabolites were assessed by liquid chromatography-mass spectrometry. Other relevant phenotypic readouts were also performed, such as heart rate, swimming activity and overall survival. To validate the specificity of our findings, we also analyzed other zebrafish genetic mutants diseased controls (i.e. nf1 CRISPants as PPGL positive controls and scn1a-mutant as non-PPGL negative genetic controls). Finally, we performed an RNA-sequencing assay from sdhb-CRISPant and controls larvae. Results: Sdhb CRISPants exhibit increased heart rates, reduced swimming activity and premature death. In whole fish extracts, normetanephrine (NM), metanephrine (MN), and dopamine (DA) levels were about three times higher in sdhb CRISPants than in control larvae. In the bathing medium, NM and MN were also significantly elevated, along with 3-MT. We confirmed the specificity of our findings by showing that nf1-mutant larvae exhibit similar catecholamine levels but also show an adrenergic phenotype typical of cluster 2 variants of PPGLs. In contrast, scn1a-mutant larvae, despite suffering from severe epileptic encephalopathy associated with reduced lifespan, did not display elevated catecholamine levels. Complementary metabolic profiling showed that sdhb CRISPants depict a clear Complex II dysfunction signature, characterized by elevated succinate, lactate, NAD+, along with reduced aspartate and glutamate levels. Additionally, RNA-sequencing revealed downregulation of sdhb and fads2, and upregulation of genes involved in the hypoxia response, angiogenesis, stress response, and glycolysis in sdhb CRISPants compared to Cas9-control larvae. Conclusion: In this work, we validated the relevance of F0 loss-of-function zebrafish models (CRISPant) to study the pathogenicity of PPGL-causing genetic variants in vivo. We present the first in vivo model of sdhb loss-of-function with oversecretion of free catecholamines, a key clinical hallmark of PPGLs in humans. We also show that other pathological molecular hallmarks are found increased such as Krebs cycle metabolites and relevant phenotypic readouts (tachycardia, reduced lifespan, and consistent transcriptomic dysregulations). These findings suggest that zebrafish is a relevant physiopathologic model for studying PPGLs and their associated genetic etiologies and opens the door for streamlining preclinical precision medicine approaches for PPGLs.

Dataset Information

Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

Publications

Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

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