In solid tumors, neoplastic cells grow in contact with the so-called tumor microenvironment. The interaction between tumor cells and the microenvironment causes reciprocal metabolic reprogramming and favorable conditions for tumor growth and metastatic spread. To obtain an experimental model resembling the in vivo conditions of the succinate dehydrogenase B subunit (SDHB)-mutated paragangliomas (PGLs), we evaluated the effects of SDHB silencing on metabolism and proliferation in the human neuroblastoma cell line (SK-N-AS), cultured alone or in association with human fibroblasts. Silencing caused a 70% decrease in protein expression, an almost complete loss of the complex specific enzymatic activity, and a significant increase in HIF1α and HIF2α expression; it thus resembled the in vivo tumor cell phenotype. When compared with WT SK-N-AS cells, SDHB-silenced cells showed an altered metabolism characterized by an unexpected significant decrease in glucose uptake and an increase in lactate uptake. Moreover, silenced cells exhibited a significant increase in cell proliferation and metalloproteinase activity. When co-cultured with human fibroblasts, control cells displayed a significant decrease in glucose uptake and a significant increase in cell proliferation as compared with their mono-cultured counterparts. These effects were even more evident in co-cultured silenced cells, with a 70% decrease in glucose uptake and a 92% increase in cell proliferation as compared to their mono-cultured counterparts. The present data indicate for the first time, to our knowledge, that SDHB impairment causes metabolic and functional derangement of neural-crest-derived tumor cells and that the microenvironment, here represented by fibroblasts, strongly affects their tumor metabolism and growth capacity.
You are looking at 1 - 9 of 9 items for
- Author: Susan Richter x
- Refine by Access: Content accessible to me x
Elena Rapizzi, Rossella Fucci, Elisa Giannoni, Letizia Canu, Susan Richter, Paolo Cirri, and Massimo Mannelli
Susan Richter, Timothy J Garrett, Nicole Bechmann, Roderick J Clifton-Bligh, and Hans K Ghayee
Metabolites represent the highest layer of biological information. Their diverse chemical nature enables networks of chemical reactions that are critical for maintaining life by providing energy and building blocks. Quantification by targeted and untargeted analytical methods using either mass spectrometry or nuclear magnetic resonance spectroscopy has been applied to pheochromocytoma/paraganglioma (PPGL) with the long-term goal to improve diagnosis and therapy. PPGLs have unique features that provide useful biomarkers and clues for targeted treatments. First, high production rates of catecholamines and metanephrines allow for specific and sensitive detection of the disease in plasma or urine. Secondly, PPGLs are associated with heritable pathogenic variants (PVs) in around 40% of cases, many of which occur in genes encoding enzymes, such as succinate dehydrogenase (SDH) and fumarate hydratase (FH). These genetic aberrations lead to the overproduction of oncometabolites succinate or fumarate, respectively, and are detectable in tumors and blood. Such metabolic dysregulation can be exploited diagnostically, with the aim to ensure appropriate interpretation of gene variants, especially those with unknown significance, and facilitate early tumor detection through regular patient follow-up. Furthermore, SDHx and FH PV alter cellular pathways, including DNA hypermethylation, hypoxia signaling, redox homeostasis, DNA repair, calcium signaling, kinase cascades, and central carbon metabolism. Pharmacological interventions targeted toward such features have the potential to uncover treatments against metastatic PPGL, around 50% of which are associated with germline PV in SDHx. With the availability of omics technologies for all layers of biological information, personalized diagnostics and treatment is in close reach.
Martin Ullrich, Josephine Liers, Mirko Peitzsch, Anja Feldmann, Ralf Bergmann, Ulrich Sommer, Susan Richter, Stefan R Bornstein, Michael Bachmann, Graeme Eisenhofer, Christian G Ziegler, and Jens Pietzsch
Somatostatin receptor-targeting endoradiotherapy offers potential for treating metastatic pheochromocytomas and paragangliomas, an approach likely to benefit from combination radiosensitization therapy. To provide reliable preclinical in vivo models of metastatic disease, this study characterized the metastatic spread of luciferase-expressing mouse pheochromocytoma (MPC) cells in mouse strains with different immunologic conditions. Bioluminescence imaging showed that, in contrast to subcutaneous non-metastatic engraftment of luciferase-expressing MPC cells in NMRI-nude mice, intravenous cell injection provided only suboptimal metastatic spread in both NMRI-nude mice and hairless SCID (SHO) mice. Treatment of NMRI-nude mice with anti-Asialo GM1 serum enhanced metastatic spread due to substantial depletion of natural killer (NK) cells. However, reproducible metastatic spread was only observed in NK cell-defective SCID/beige mice and in hairless immunocompetent SKH1 mice bearing disseminated or liver metastases, respectively. Liquid chromatography tandem mass spectrometry of urine samples showed that subcutaneous and metastasized tumor models exhibit comparable renal monoamine excretion profiles characterized by increasing urinary dopamine, 3-methoxytyramine, norepinephrine and normetanephrine. Metastases-related epinephrine and metanephrine were only detectable in SCID/beige mice. Positron emission tomography and immunohistochemistry revealed that all metastases maintained somatostatin receptor-specific radiotracer uptake and immunoreactivity, respectively. In conclusion, we demonstrate that intravenous injection of luciferase-expressing MPC cells into SCID/beige and SKH1 mice provides reproducible and clinically relevant spread of catecholamine-producing and somatostatin receptor-positive metastases. These standardized preclinical models allow for precise monitoring of disease progression and should facilitate further investigations on theranostic approaches against metastatic pheochromocytomas and paragangliomas.
Vanessa D'Antongiovanni, Serena Martinelli, Susan Richter, Letizia Canu, Daniele Guasti, Tommaso Mello, Paolo Romagnoli, Karel Pacak, Graeme Eisenhofer, Massimo Mannelli, and Elena Rapizzi
Pheochromocytomas (Pheos) and paragangliomas (PGLs) are neuroendocrine tumors. Approximately 30–40% of Pheos/PGLs are due to germline mutations in one of the susceptibility genes, including those encoding the succinate dehydrogenase subunits A-D (SDHA-D). Up to 2/3 of patients affected by SDHB mutated Pheo/PGL develop metastatic disease with no successful cure at present. Here, for the first time, we evaluated the effects of SDHB silencing in a three dimension (3D) culture using spheroids of a mouse Pheo cell line silenced or not (wild type/wt/control) for the SDHB subunit. We investigated the role of the microenvironment on spheroid growth and migration/invasion by co-culturing SDHB-silenced or wt spheroids with primary cancer-activated fibroblasts (CAFs). When spheroids were co-cultured with fibroblasts, SDHB-silenced cells showed a significant increase in matrigel invasion as demonstrated by the computation of the migratory areas (P < 0.001). Moreover, cells detaching from the SDHB-silenced spheroids moved collectively, unlike the cells of wt spheroids that moved individually. Additionally, SDHB-silenced spheroids developed long filamentous formations along which clusters of cells migrated far away from the spheroid, whereas these structures were not present in wt spheroids. We found that lactate, largely secreted by CAFs, plays a specific role in promoting migration only of SDHB-silenced cells. In this study, we demonstrated that SDHB silencing per se increases tumor cell migration/invasion and that microenvironment, as represented by CAFs, plays a pivotal role in enhancing collective migration/invasion in Pheo SDHB-silenced tumor cells, suggesting their role in increasing the tumor metastasizing potential.
Margo Dona, Selma Waaijers, Susan Richter, Graeme Eisenhofer, Jeroen Korving, Sarah M Kamel, Jeroen Bakkers, Elena Rapizzi, Richard J Rodenburg, Jan Zethof, Marnix Gorissen, Gert Flik, Peter M T Deen, and Henri J L M Timmers
Pheochromocytomas and paragangliomas (PPGLs) caused by mutations in the B-subunit of the succinate dehydrogenase (SDHB) have the highest metastatic rate among PPGLs, and effective systemic therapy is lacking. To unravel underlying pathogenic mechanisms, and to evaluate therapeutic strategies, suitable in vivo models are needed. The available systemic Sdhb knock-out mice cannot model the human PPGL phenotype: heterozygous Sdhb mice lack a disease phenotype, and homozygous Sdhb mice are embryonically lethal. Using CRISPR/cas9 technology, we introduced a protein-truncating germline lesion into the zebrafish sdhb gene. Heterozygous sdhb mutants were viable and displayed no obvious morphological or developmental defects. Homozygous sdhb larvae were viable, but exhibited a decreased lifespan. Morphological analysis revealed incompletely or non-inflated swim bladders in homozygous sdhb mutants at day 6. Although no differences in number and ultrastructure of the mitochondria were observed. Clear defects in energy metabolism and swimming behavior were observed in homozygous sdhb mutant larvae. Functional and metabolomic analyses revealed decreased mitochondrial complex 2 activity and significant succinate accumulation in the homozygous sdhb mutant larvae, mimicking the metabolic effects observed in SDHB-associated PPGLs. This is the first study to present a vertebrate animal model that mimics metabolic effects of SDHB-associated PPGLs. This model will be useful in unraveling pathomechanisms behind SDHB-associated PPGLs. We can now study the metabolic effects of sdhb disruption during different developmental stages and develop screening assays to identify novel therapeutic targets in vivo. Besides oncological syndromes, our model might also be useful for pediatric mitochondrial disease caused by loss of the SDHB gene.
Susan Richter, Bei Qiu, Mirthe Ghering, Carola Kunath, Georgiana Constantinescu, Charlotte Luths, Christina Pamporaki, Nicole Bechmann, Leah Meuter, Aleksandra Kwapiszewska, Timo Deutschbein, Svenja Nölting, Mirko Peitzsch, Mercedes Robledo, Aleksander Prejbisz, Karel Pacak, Volker Gudziol, Henri J L M Timmers, and Graeme Eisenhofer
Head and neck paragangliomas (HNPGLs) are tumors of parasympathetic origin that occur at variable locations and are often secondary to germline mutations in succinate dehydrogenase (SDH) subunit genes. Occasionally, these tumors produce catecholamines. Here, we assessed whether different locations of HNPGLs relate to the presence of SDHx mutations, catecholamine production and other presentations. In this multicenter study, we collected clinical and biochemical data from 244 patients with HNPGLs and 71 patients without HNPGLs. We clarified that jugulotympanic HNPGLs have distinct features. In particular, 88% of jugulotympanic HNPGLs arose in women, among whom only 24% occurred due to SDHx mutations compared to 55% in men. Jugulotympanic HNPGLs were also rarely bilateral, were of a smaller size and were less often metastatic compared to carotid body and vagal HNPGLs. Furthermore, we showed that plasma concentrations of methoxytyramine (MTY) were higher (P < 0.0001) in patients with HNPGL than without HNPGL, whereas plasma normetanephrine did not differ. Only 3.7% of patients showed strong increases in plasma normetanephrine. Plasma MTY was positively related to tumor size but did not relate to the presence of SDHx mutations or tumor location. Our findings confirm that increases in plasma MTY represent the main catecholamine-related biochemical feature of patients with HNPGLs. We expect that more sensitive analytical methods will make biochemical testing of HNPGLs more practical in the future and enable more than the current 30% of patients to be identified with dopamine-producing HNPGLs. The sex-dependent differences in the development of HNPGLs may have relevance to the diagnosis, management and outcomes of these tumors.
Trisha Dwight, Edward Kim, Karine Bastard, Diana E Benn, Graeme Eisenhofer, Susan Richter, Massimo Mannelli, Elena Rapizzi, Aleksander Prejbisz, Mariola Pęczkowska, Karel Pacak, and Roderick Clifton-Bligh
Mosaic or somatic EPAS1 mutations are associated with a range of phenotypes including pheochromocytoma and/or paraganglioma (PPGL), polycythemia and somatostatinoma. The pathogenic potential of germline EPAS1 variants however is not well understood. We report a number of germline EPAS1 variants occurring in patients with PPGL, including a novel variant c.739C>A (p.Arg247Ser); a previously described variant c.1121T>A (p.Phe374Tyr); several rare variants, c.581A>G (p.His194Arg), c.2353C>A (p.Pro785Thr) and c.2365A>G (p.Ile789Val); a common variant c.2296A>C (p.Thr766Pro). We performed detailed functional studies to understand their pathogenic role in PPGL. In transient transfection studies, EPAS1/HIF-2α p.Arg247Ser, p.Phe374Tyr and p.Pro785Thr were all stable in normoxia. In co-immunoprecipitation assays, only the novel variant p.Arg247Ser showed diminished interaction with pVHL. A direct interaction between HIF-2α Arg247 and pVHL was confirmed in structural models. Transactivation was assessed by means of a HRE-containing reporter gene in transiently transfected cells, and significantly higher reporter activity was only observed with EPAS1/HIF-2α p.Phe374Tyr and p.Pro785Thr. In conclusion, three germline EPAS1 variants (c.739C>A (p.Arg247Ser), c.1121T>A (p.Phe374Tyr) and c.2353C>A (p.Pro785Thr)) all have some functional features in common with somatic activating mutations. Our findings suggest that these three germline variants are hypermorphic alleles that may act as modifiers to the expression of PPGLs.
Katharina Wang, Ina Schütze, Sebastian Gulde, Nicole Bechmann, Susan Richter, Jana Helm, Michael Lauseker, Julian Maurer, Astrid Reul, Gerald Spoettl, Barbara Klink, Doreen William, Thomas Knösel, Juliane Friemel, Michel Bihl, Achim Weber, Maria Fankhauser, Laura Schober, Diana Vetter, Martina Broglie Däppen, Christian G Ziegler, Martin Ullrich, Jens Pietzsch, Stefan R Bornstein, Christian Lottspeich, Matthias Kroiss, Martin Fassnacht, Vera Ursula Julia Wenter, Roland Ladurner, Constanze Hantel, Martin Reincke, Graeme Eisenhofer, Ashley B Grossman, Karel Pacak, Felix Beuschlein, Christoph J Auernhammer, Natalia S Pellegata, and Svenja Nölting
Aggressive pheochromocytomas and paragangliomas (PPGLs) are difficult to treat, and molecular targeting is being increasingly considered, but with variable results. This study investigates established and novel molecular-targeted drugs and chemotherapeutic agents for the treatment of PPGLs in human primary cultures and murine cell line spheroids. In PPGLs from 33 patients, including 7 metastatic PPGLs, we identified germline or somatic driver mutations in 79% of cases, allowing us to assess potential differences in drug responsivity between pseudohypoxia-associated cluster 1-related (n = 10) and kinase signaling-associated cluster 2-related (n = 14) PPGL primary cultures. Single anti-cancer drugs were either more effective in cluster 1 (cabozantinib, selpercatinib, and 5-FU) or similarly effective in both clusters (everolimus, sunitinib, alpelisib, trametinib, niraparib, entinostat, gemcitabine, AR-A014418, and high-dose zoledronic acid). High-dose estrogen and low-dose zoledronic acid were the only single substances more effective in cluster 2. Neither cluster 1- nor cluster 2-related patient primary cultures responded to HIF-2a inhibitors, temozolomide, dabrafenib, or octreotide. We showed particular efficacy of targeted combination treatments (cabozantinib/everolimus, alpelisib/everolimus, alpelisib/trametinib) in both clusters, with higher efficacy of some targeted combinations in cluster 2 and overall synergistic effects (cabozantinib/everolimus, alpelisib/trametinib) or synergistic effects in cluster 2 (alpelisib/everolimus). Cabozantinib/everolimus combination therapy, gemcitabine, and high-dose zoledronic acid appear to be promising treatment options with particularly high efficacy in SDHB-mutant and metastatic tumors. In conclusion, only minor differences regarding drug responsivity were found between cluster 1 and cluster 2: some single anti-cancer drugs were more effective in cluster 1 and some targeted combination treatments were more effective in cluster 2.
Nicole Bechmann, Mats Leif Moskopp, Martin Ullrich, Bruna Calsina, Pål William Wallace, Susan Richter, Markus Friedemann, Katharina Langton, Stephanie M J Fliedner, Henri J L M Timmers, Svenja Nölting, Felix Beuschlein, Martin Fassnacht, Aleksander Prejbisz, Karel Pacak, Hans K Ghayee, Stefan R Bornstein, Peter Dieterich, Jens Pietzsch, Ben Wielockx, Mercedes Robledo, Nan Qin, and Graeme Eisenhofer
Mutations that drive the stabilization of hypoxia inducible factor 2α (HIF2α) and downstream pseudohypoxic signaling are known to predispose to the development of pheochromocytomas and paragangliomas (PPGLs). However, any role of HIF2α in predisposition to metastatic disease remains unclear. To assess such a role we combined gene-manipulations in pheochromocytoma cell lines with retrospective analyses of patient data and gene expression profiling in tumor specimens. Among 425 patients with PPGLs identified with mutations in tumor-susceptibility genes, those with tumors due to activation of pseudohypoxic pathways had a higher frequency of metastatic disease than those with tumors due to activation of kinase-signaling pathways, even without inclusion of patients with mutations in SDHB (18.6% vs 4.3% in, P < 0.0001). Three out of nine (33%) patients with gain-of-function mutations in HIF2α had metastatic disease. In cell line studies, elevated expression of HIF2α enhanced cell proliferation and led to increased migration and invasion capacity. Moreover, HIF2α expression in HIF2α-deficient cells resulted in increased cell motility, diffuse cluster formation and emergence of pseudopodia indicating changes in cell adhesion and cytoskeletal remodeling. In a mouse liver metastasis model, Hif2a enhanced the metastatic load. Transcriptomics data revealed alterations in focal adhesion and extracellular matrix–receptor interactions in HIF2α-mutated PPGLs. Our translational findings demonstrate that HIF2α supports pro-metastatic behavior in PPGLs, though other factors remain critical for subsequent transition to metastasis. We identified LAMB1 and COL4A2 as new potential therapeutic targets for HIF2α-driven PPGLs. Identified HIF2α downstream targets might open a new therapeutic window for aggressive HIF2α-expressing tumors.