Characterisation of an Adult Zebrafish Model for SDHB-Associated Phaeochromocytomas and Paragangliomas
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SUBMITTER: Miltenburg J
PROVIDER: S-EPMC11241774 | biostudies-literature | 2024 Jul
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC7139890 | biostudies-literature
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.
2025-08-20 | GSE300914 | GEO
Project description:Endothelial PAS domain-containing protein 1 (EPAS1) is an oxygen-sensitive component of the hypoxia-inducible factors (HIFs) having reported implications in many cancers by inducing a pseudo-hypoxic microenvironment. However, the molecular dysregulation and clinical significance of EPAS1 has never been investigated in depth in phaeochromocytomas/paragangliomas. This study aims to identify EPAS1 mutations and alterations in DNA copy number, mRNA and protein expression in patients with phaeochromocytomas/paragangliomas. The association of molecular dysregulations of EPAS1 with clinicopathological factors in phaeochromocytomas and paragangliomas were also analysed. High-resolution melt-curve analysis followed by Sanger sequencing was used to detect mutations in EPAS1. EPAS1 DNA number changes and mRNA expressions were examined by polymerase chain reaction (PCR). Immunofluorescence assay was used to study EPAS1 protein expression. In phaeochromocytomas, 12% (n = 7/57) of patients had mutations in the EPAS1 sequence, which includes two novel mutations (c.1091A > T; p.Lys364Met and c.1129A > T; p.Ser377Cys). Contrastingly, in paragangliomas, 7% (n = 1/14) of patients had EPAS1 mutations and only the c.1091A > T; p.Lys364Met mutation was detected. In silico analysis revealed that the p.Lys364Met mutation has pathological potential based on the functionality of the protein, whereas the p.Ser377Cys mutation was predicted to be neutral or tolerated. The majority of the patients had EPAS1 DNA amplification (79%; n = 56/71) and 53% (n = 24/45) patients shown mRNA overexpression. Most of the patients with EPAS1 mutations exhibited aberrant DNA changes, mRNA and protein overexpression. In addition, these alterations of EPAS1 were associated with tumour weight and location. Thus, the molecular dysregulation of EPAS1 could play crucial roles in the pathogenesis of phaeochromocytomas and paragangliomas.
| S-EPMC7693385 | biostudies-literature
Project description:Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background in over one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and several other tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlighting the importance of identifying SDHx mutations for patient management. Genetic variants of unknown significance, where implications for the patient and family members are unclear, are a problem for interpretation. For such cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB (SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatography-mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions provide an alternative method. Here, we compare SDHB-IHC with metabolite profiling in 189 tumours from 187 PPGL patients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to establish predictive models for interpreting metabolite data. Metabolite profiling showed higher diagnostic specificity compared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite profiles improved predictive ability over that of the SFR, in particular for hard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, the combination of metabolite profiling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classified all but one of the false negatives from metabolite profiling strategies, while metabolite profiling correctly classified all but one of the false negatives/positives from SDHB-IHC. From 186 tumours with confirmed status of SDHx variant pathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benefits of both strategies for patient management. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
| S-EPMC7548960 | biostudies-literature
Project description:AimTo develop and internally validate a novel predictive model for SDHB mutations in pheochromocytomas and retroperitoneal paragangliomas (PPGLs).MethodsClinical data of patients with PPGLs who presented to Peking Union Medical College Hospital from 2013 to 2022 and underwent genetic testing were retrospectively collected. Variables were screened by backward stepwise and clinical significance and were used to construct multivariable logistic models in 50 newly generated datasets after the multiple imputation. Bootstrapping was used for internal validation. A corresponding nomogram was generated based on the model. Sensitivity analyses were also performed.ResultsA total of 556 patients with PPGLs were included, of which 99 had a germline SDHB mutation. The prediction model revealed that younger age of onset [Odds ratio (OR): 0.93, 95% CI: 0.91-0.95], synchronous metastasis (OR: 6.43, 95% CI: 2.62-15.80), multiple lesion (OR: 0.22, 95% CI: 0.09-0.54), retroperitoneal origin (OR: 5.72, 95% CI: 3.13-10.47), negative 131I-meta-iodobenzylguanidine (MIBG) (OR: 0.34, 95% CI: 0.15-0.73), positive octreotide scintigraphy (OR: 3.24, 95% CI: 1.25-8.43), elevated 24h urinary dopamine (DA) (OR: 1.72, 95% CI: 0.93-3.17), NE secretory type (OR: 2.83, 95% CI: 1.22- 6.59), normal secretory function (OR: 3.04, 95% CI: 1.04-8.85) and larger tumor size (OR: 1.09, 95% CI: 0.99-1.20) were predictors of SDHB mutations in PPGLs, and showed good and stable predictive performance with a mean area under the ROC curve (AUC) of 0.865 and coefficient of variation of 2.2%.ConclusionsThis study provided a novel and useful tool for predicting SDHB mutations by integrating easily obtained clinical data. It may help clinicians select suitable genetic testing methods and make appropriate clinical decisions for these high-risk patients.
| S-EPMC10764617 | biostudies-literature
Project description:Rapid generation of a sdhb loss-of-function zebrafish model for secreting pheochromocytomas and paragangliomas
| PRJNA1282665 | ENA
Project description:Germline mutations in the succinate dehydrogenase (SDHA, SDHB, SDHC, SDHD, SDHAF2) or Von Hippel-Lindau (VHL) genes cause hereditary paraganglioma/pheochromocytoma. While SDHB (1p36) and VHL (3p25) are associated with autosomal dominant disease, SDHD (11q23) and SDHAF2 (11q13) show a remarkable parent-of-origin effect whereby tumor formation is almost completely dependent on paternal transmission of the mutant allele. Loss of the entire maternal copy of chromosome 11 occurs frequently in SDHD-linked tumors, and has been suggested to be the basis for this typical inheritance pattern.Using fluorescent in situ hybridization, microsatellite marker and SNP array analysis, we demonstrate that loss of the entire copy of chromosome 11 is also frequent in SDHAF2-related PGLs, occurring in 89% of tumors. Analysis of two imprinted differentially methylated regions (DMR) in 11p15, H19-DMR and KvDMR, showed that this loss always affected the maternal copy of chromosome 11. Likewise, loss of maternal chromosome 11p15 was demonstrated in 85% of SDHD and 75% of VHL-related PGLs/PCCs. By contrast, both copies of chromosome 11 were found to be retained in 62% of SDHB-mutated PGLs/PCCs, while only 31% showed loss of maternal chromosome 11p15. Genome-wide copy number analysis revealed frequent loss of 1p in SDHB mutant tumors and show greater genomic instability compared to SDHD and SDHAF2.These results show that loss of the entire copy of maternal chromosome 11 is a highly specific and statistically significant event in SDHAF2, SDHD and VHL-related PGLs/PCCs, but is less significant in SDHB-mutated tumors, suggesting that these tumors have a distinct genetic etiology.
| S-EPMC5362423 | biostudies-literature
Project description: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.
| S-EPMC9524698 | biostudies-literature
Project description:The hypoxic response underlies the pathogenesis and malignant behavior of PCC/PGL. Regulation of PD-1 receptor-ligand signaling, a therapeutically actionable driver of the anti-tumor immune response, is a hypoxic-driven trait across malignancies. We evaluated the prognostic role of PD ligands in association with biomarkers of hypoxia and angiogenesis in patients with PCC/PGL. Tissue microarrays sections including consecutive cases diagnosed between 1983-2011 were stained for PD-L1 and 2, hypoxia inducible factor 1a (Hif-1a), Carbonic Anhydrase IX (CaIX), Vascular Endothelial Growth Factor-A (VEGF-A). We explored the biologic significance of PD ligands expression using gene set enrichment analysis (GSEA) on The Cancer Genome Atlas (TCGA) for PCC/PGL (n = 184). In total, 100 patients, 10% malignant, 64% PCC, 29% familial with median tumor size of 4.7 cm (range 1-14) were included. Median follow-up was 4.7 y. We found PD-L1 expression in 18% of PCC/PGL, which was independent of adverse pathological features including capsular (CI), vascular invasion (VI), necrosis (N) and expression of biomarkers of hypoxia. PD-L2 expression (16%) strongly correlated with CI, VI, N and malignant behavior (p < 0.05) and was associated with stronger Hif-1a and CaIX immunolabeling (p < 0.01). PD-L2 was predictive of shorter survival (162 versus 309 months, HR 3.1 95%CI 1.1-9.2, p = 0.02). GSEA on TGCA samples confirmed enrichment of transcripts involved in hypoxia and anti-cancer immunity. We report for the first time PD ligands expression in PCC/PGL with a distinctive prognostic, clinico-pathologic and immuno-biologic role. These findings support a potential therapeutic role for PD-1/PD-L1 targeted checkpoint inhibitors in these tumors. KEY MESSAGE The molecular mechanisms underlying immune evasion in malignant phaeochromocytomas and paragangliomas (PCC/PGL) are poorly understood. This study demonstrates for the first time a distinctive immune-biologic and prognostic role of programmed death ligands 1 and 2 (PD-L1, PD-L2), two actionable drivers of the anti-cancer immune response. RNA-sequencing of tumor tissues reveals enrichment of transcripts relating to hypoxia and immune-exhaustion to explain the adverse clinical course observed in PD-L2 overexpressing tumors. These findings provide a rationale for the development of anti PD-1 therapies in malignant PCC/PGL.
| S-EPMC5674959 | biostudies-literature
Project description:Urinary bladder paraganglioma (paraganglioma) is a rare tumor of chromaffin cells of the sympathetic system of the urinary bladder wall. We studied 14 cases of this entity and investigated the usefulness of SDHB protein staining by immunohistochemistry (IHC) as a diagnostic tool to identify patients with bladder paragangliomas that could be associated with SDHB gene mutations, as these patients have a more aggressive disease. Eleven tumors from these patients were stained by IHC. Six of 11 tumors were negative for SDHB staining by IHC with no cytoplasmic staining in tumor cells when compared with normal tissues. Five of these 6 negative cases were confirmed to be positive for germline SDHB mutations. One case showed negative staining and no germline SDHB mutation; however, further investigation of the tumor revealed a somatic SDHB gene deletion. The remaining 5 cases showed strong cytoplasmic staining, but they were negative for the presence of SDHB mutation. They were found to be either sporadic tumors or part of von Hippel-Lindau syndrome. Staining for SDHA was positive in all cases. Our study confirms that there is very good correlation between the presence of an SDHB mutation, whether germline or sporadic, and negative SDHB IHC staining in urinary bladder paragangliomas, and this is the first study to demonstrate that somatic mutations can be recognized by IHC staining.
| S-EPMC4606469 | biostudies-literature