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Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells

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Abstract

Pheochromocytomas and paragangliomas (PGLs) due to mutations of succinate dehydrogenase (SDH) B, a subunit of the SDH complex with a role in the Krebs cycle and the respiratory chain, tend to be larger at diagnosis and more prone to metastatic disease than other tumors. This presentation contrasts with the behavior of some cell line models of SDHB impairment, which show reduced growth compared to wild type. We hypothesize that reduced growth of SDHB-impaired monolayer culture models might reflect lack of support from sources within the tumor microenvironment. The present study therefore investigates how the microenvironment, modeled here by fibroblast co-culture, modulates cell metabolism, growth and invasion in an Sdhb-impaired mouse pheochromocytoma cell line. We employed two different constructs of short hairpin RNA to knockdown Sdhb and compared growth in a monolayer with and without fibroblast co-culture. Sdhb-silenced cells showed functional impairment of SDH with elevated succinate to fumarate ratio and decreased oxidative capacity. Cell growth was delayed with an increase in doubling time of 2 h or 20 h. Clonogenic cell survival and viability, on the other hand, were either unchanged or increased compared to control. In standard monolayer culture, no differences in pro-metastatic features were present. Co-culture with primary mouse fibroblast reversed the difference of proliferation between control and Sdhb knockdown but was unable to significantly influence invasiveness under these culture conditions. Metabolic studies identified that lactate secreted by fibroblasts was taken up preferentially by Sdhb-silenced cells. In summary, the present study identified a potential role for the tumor microenvironment in influencing phenotypic features of SDHB-mutated PGLs, providing a basis for the use of therapies targeted towards the tumor microenvironment.

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References

  • Aspuria PJ, Lunt SY, Varemo L, Vergnes L, Gozo M, Beach JA, Salumbides B, Reue K, Wiedemeyer WR, Nielsen J, Karlan BY, Orsulic S (2014) Succinate dehydrogenase inhibition leads to epithelial-mesenchymal transition and reprogrammed carbon metabolism. Cancer Metab 2:21

    Article  Google Scholar 

  • Cardaci S, Zheng L, MacKay G, van den Broek NJ, MacKenzie ED, Nixon C, Stevenson D, Tumanov S, Bulusu V, Kamphorst JJ, Vazquez A, Fleming S, Schiavi F, Kalna G, Blyth K, Strathdee D, Gottlieb E (2015) Pyruvate carboxylation enables growth of SDH-deficient cells by supporting aspartate biosynthesis. Nat Cell Biol 17:1317–1326

    Article  CAS  Google Scholar 

  • Cervera AM, Apostolova N, Crespo FL, Mata M, McCreath KJ (2008) Cells silenced for SDHB expression display characteristic features of the tumor phenotype. Cancer Res 68:4058–4067

    Article  CAS  Google Scholar 

  • Chiarugi P, Cirri P (2016) Metabolic exchanges within tumor microenvironment. Cancer Lett 380:272–280

    Article  CAS  Google Scholar 

  • D’Antongiovanni V, Martinelli S, Richter S, Canu L, Guasti D, Mello T, Romagnoli P, Pacak K, Eisenhofer G, Mannelli M, Rapizzi E (2017) The microenvironment induces collective migration in SDHB-silenced mouse pheochromocytoma spheroids. Endocr Relat Cancer 24:555–564

    Article  Google Scholar 

  • Daubner SC, Le T, Wang S (2011) Tyrosine hydroxylase and regulation of dopamine synthesis. Arch Biochem Biophys 508:1–12

    Article  CAS  Google Scholar 

  • Eisenhofer G, Goldstein DS, Stull R, Keiser HR, Sunderland T, Murphy DL, Kopin IJ (1986) Simultaneous liquid-chromatographic determination of 3,4-dihydroxyphenylglycol, catecholamines, and 3,4-dihydroxyphenylalanine in plasma, and their responses to inhibition of monoamine oxidase. Clin Chem 32:2030–2033

    CAS  PubMed  Google Scholar 

  • Eisenhofer G, Lenders JW, Siegert G, Bornstein SR, Friberg P, Milosevic D, Mannelli M, Linehan WM, Adams K, Timmers HJ, Pacak K (2012) Plasma methoxytyramine: a novel biomarker of metastatic pheochromocytoma and paraganglioma in relation to established risk factors of tumour size, location and SDHB mutation status. Eur J Cancer 48:1739–1749

    Article  CAS  Google Scholar 

  • Favier J, Amar L, Gimenez-Roqueplo AP (2015) Paraganglioma and phaeochromocytoma: from genetics to personalized medicine. Nat Rev Endocrinol 11:101–111

    Article  CAS  Google Scholar 

  • Fiaschi T, Marini A, Giannoni E, Taddei ML, Gandellini P, De Donatis A, Lanciotti M, Serni S, Cirri P, Chiarugi P (2012) Reciprocal metabolic reprogramming through lactate shuttle coordinately influences tumor-stroma interplay. Cancer Res 72:5130–5140

    Article  CAS  Google Scholar 

  • Ghayee HK, Bhagwandin VJ, Stastny V, Click A, Ding LH, Mizrachi D, Zou YS, Chari R, Lam WL, Bachoo RM, Smith AL, Story MD, Sidhu S, Robinson BG, Nwariaku FE, Gazdar AF, Auchus RJ, Shay JW (2013) Progenitor cell line (hPheo1) derived from a human pheochromocytoma tumor. PLoS One 8:e65624

    Article  CAS  Google Scholar 

  • Gimenez-Roqueplo AP, Favier J, Rustin P, Rieubland C, Crespin M, Nau V, Khau Van Kien P, Corvol P, Plouin PF, Jeunemaitre X (2003) Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas. Cancer Res 63:5615–5621

    CAS  PubMed  Google Scholar 

  • Guzy RD, Sharma B, Bell E, Chandel NS, Schumacker PT (2008) Loss of the SdhB, but not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis. Mol Cell Biol 28:718–731

    Article  CAS  Google Scholar 

  • Hanahan D, Coussens LM (2012) Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21:309–322

    Article  CAS  Google Scholar 

  • Imperiale A, Moussallieh FM, Sebag F, Brunaud L, Barlier A, Elbayed K, Bachellier P, Goichot B, Pacak K, Namer IJ, Taieb D (2013) A new specific succinate-glutamate metabolomic hallmark in sdhx-related paragangliomas. PLoS One 8:e80539

    Article  Google Scholar 

  • Jochmanova I, Pacak K (2016) Pheochromocytoma: the first metabolic endocrine cancer. Clin Cancer Res: Off J Am Assoc Cancer Res 22:5001–5011

    Article  CAS  Google Scholar 

  • King KS, Prodanov T, Kantorovich V, Fojo T, Hewitt JK, Zacharin M, Wesley R, Lodish M, Raygada M, Gimenez-Roqueplo AP, McCormack S, Eisenhofer G, Milosevic D, Kebebew E, Stratakis CA, Pacak K (2011) Metastatic pheochromocytoma/paraganglioma related to primary tumor development in childhood or adolescence: significant link to SDHB mutations. J Clin Oncol Off J Am Soc Clin Oncol 29:4137–4142

    Article  CAS  Google Scholar 

  • Letouze E, Martinelli C, Loriot C, Burnichon N, Abermil N, Ottolenghi C, Janin M, Menara M, Nguyen AT, Benit P, Buffet A, Marcaillou C, Bertherat J, Amar L, Rustin P, De Reynies A, Gimenez-Roqueplo AP, Favier J (2013) SDH mutations establish a hypermethylator phenotype in paraganglioma. Cancer Cell 23:739–752

    Article  CAS  Google Scholar 

  • Lussey-Lepoutre C, Hollinshead KE, Ludwig C, Menara M, Morin A, Castro-Vega LJ, Parker SJ, Janin M, Martinelli C, Ottolenghi C, Metallo C, Gimenez-Roqueplo AP, Favier J, Tennant DA (2015) Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism. Nat Commun 6:8784

    Article  CAS  Google Scholar 

  • Mannelli M, Rapizzi E, Fucci R, Canu L, Ercolino T, Luconi M, Young WF Jr (2015) 15 years of paraganglioma: metabolism and pheochromocytoma/paraganglioma. Endocr Relat Cancer 22:T83–T90

    Article  CAS  Google Scholar 

  • Martiniova L, Lai EW, Elkahloun AG, Abu-Asab M, Wickremasinghe A, Solis DC, Perera SM, Huynh TT, Lubensky IA, Tischler AS, Kvetnansky R, Alesci S, Morris JC, Pacak K (2009) Characterization of an animal model of aggressive metastatic pheochromocytoma linked to a specific gene signature. Clin Exp Metastasis 26:239–250

    Article  CAS  Google Scholar 

  • Masgras I, Ciscato F, Brunati AM, Tibaldi E, Indraccolo S, Curtarello M, Chiara F, Cannino G, Papaleo E, Lambrughi M, Guzzo G, Gambalunga A, Pizzi M, Guzzardo V, Rugge M, Vuljan SE, Calabrese F, Bernardi P, Rasola A (2017) Absence of neurofibromin induces an oncogenic metabolic switch via mitochondrial ERK-mediated phosphorylation of the chaperone TRAP1. Cell Rep 18:659–672

    Article  CAS  Google Scholar 

  • Pamporaki C, Hamplova B, Peitzsch M, Prejbisz A, Beuschlein F, Timmers HJ, Fassnacht M, Klink B, Lodish M, Stratakis CA, Huebner A, Fliedner S, Robledo M, Sinnott RO, Januszewicz A, Pacak K, Eisenhofer G (2017) Characteristics of pediatric vs adult pheochromocytomas and paragangliomas. J Clin Endocrinol Metab

  • Quail DF, Joyce JA (2013) Microenvironmental regulation of tumor progression and metastasis. Nat Med 19:1423–1437

    Article  CAS  Google Scholar 

  • Rapizzi E, Ercolino T, Fucci R, Zampetti B, Felici R, Guasti D, Morandi A, Giannoni E, Giache V, Bani D, Chiarugi A, Mannelli M (2014) Succinate dehydrogenase subunit B mutations modify human neuroblastoma cell metabolism and proliferation. Hormones Cancer 5:174–184

    Article  CAS  Google Scholar 

  • Rapizzi E, Fucci R, Giannoni E, Canu L, Richter S, Cirri P, Mannelli M (2015) Role of microenvironment on neuroblastoma SK-N-AS SDHB-silenced cell metabolism and function. Endocr Relat Cancer 22:409–417

    Article  CAS  Google Scholar 

  • Richter S, Peitzsch M, Rapizzi E, Lenders JW, Qin N, de Cubas AA, Schiavi F, Rao JU, Beuschlein F, Quinkler M, Timmers HJ, Opocher G, Mannelli M, Pacak K, Robledo M, Eisenhofer G (2014) Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/paragangliomas due to succinate dehydrogenase deficiency. J Clin Endocrinol Metab 99:3903–3911

    Article  CAS  Google Scholar 

  • Saito Y, Ishii KA, Aita Y, Ikeda T, Kawakami Y, Shimano H, Hara H, Takekoshi K (2016) Loss of SDHB elevates catecholamine synthesis and secretion depending on ROS production and HIF stabilization. Neurochem Res 41:696–706

    Article  CAS  Google Scholar 

  • Ullrich M, Bergmann R, Peitzsch M, Cartellieri M, Qin N, Ehrhart-Bornstein M, Block NL, Schally AV, Pietzsch J, Eisenhofer G, Bornstein SR, Ziegler CG (2014) In vivo fluorescence imaging and urinary monoamines as surrogate biomarkers of disease progression in a mouse model of pheochromocytoma. Endocrinology 155:4149–4156

    Article  Google Scholar 

  • Wiedemann T, Peitzsch M, Qin N, Neff F, Ehrhart-Bornstein M, Eisenhofer G, Pellegata NS (2016) Morphology, biochemistry, and pathophysiology of MENX-related pheochromocytoma recapitulate the clinical features. Endocrinology 157:3157–3166

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Dr. Arthur Tischler and Dr. Lucia Martiniova, who generated mouse pheochromocytoma cells (MPCs) and mouse tumor tissue (MTT)-derived cells, respectively.

Funding

This work was supported by the Paradifference Foundation, the Fondazione Cassa di Risparmio di Pistoia e Pescia (Prot. 2016.0241/gi; to M.M.) and the Deutsche Forschungsgemeinschaft (RI 2684/1-1; CRC/TRR 205—project B10 to S.R. and project B12 to G.E.). This research was supported, in part, by the Intramural Research Program of the NIH, NICHD.

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Author notes

  1. Susan Richter and Vanessa D’Antongiovanni contributed equally to this work.

Authors and Affiliations

  1. University Hospital Carl Gustav Carus, Institute of Clinical Chemistry and Laboratory Medicine, Technische Universität Dresden, Dresden, Germany

    Susan Richter, Nicole Bechmann, David M. Poitz & Graeme Eisenhofer

  2. Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy

    Vanessa D’Antongiovanni, Serena Martinelli, Maria Riverso & Massimo Mannelli

  3. Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, USA

    Karel Pacak

  4. Department of Experimental and Clinical Medicine, University of Florence, Viale, Pieraccini 6, 50139, Florence, Italy

    Elena Rapizzi

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  1. Susan Richter
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  2. Vanessa D’Antongiovanni
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  3. Serena Martinelli
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  4. Nicole Bechmann
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Correspondence to Elena Rapizzi.

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Richter, S., D’Antongiovanni, V., Martinelli, S. et al. Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells. Cell Tissue Res 374, 473–485 (2018). https://doi.org/10.1007/s00441-018-2907-x

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  • DOI: https://doi.org/10.1007/s00441-018-2907-x

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