Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling

Alessandro Cama, Fabio Verginelli, Lavinia Vittoria Lotti, Francesco Napolitano, Annalisa Morgano, Andria D'Orazio, Michele Vacca, Silvia Perconti, Felice Pepe, Federico Romani, Francesca Vitullo, Filippo Di Lella, Rosa Visone, Massimo Mannelli, Hartmut P H Neumann, Giancarlo Raiconi, Carlo Paties, Antonio Moschetta, Roberto Tagliaferri, Angelo Veronese & 2 others Mario Sanna, Renato Mariani-Costantini

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The NOTCH signaling pathway was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.

Original languageEnglish (US)
Pages (from-to)575-594
Number of pages20
JournalActa Neuropathologica
Volume126
Issue number4
DOIs
StatePublished - 2013

Fingerprint

Paraganglioma
Epigenomics
Neck
MicroRNAs
Head
Neoplasms
Neuroglia
Genes
Down-Regulation
Organoids
Neuroendocrine Cells
Gene Dosage
Germ-Line Mutation
Neuroblastoma
Blood Vessels
Proteins
Cell Death
Up-Regulation
Endothelial Cells
Cell Culture Techniques

Keywords

  • CNV
  • Head and neck
  • MicroRNA
  • NOTCH signaling
  • Paraganglioma
  • Paraganglioma cell culture

ASJC Scopus subject areas

  • Clinical Neurology
  • Pathology and Forensic Medicine
  • Cellular and Molecular Neuroscience

Cite this

Cama, A., Verginelli, F., Lotti, L. V., Napolitano, F., Morgano, A., D'Orazio, A., ... Mariani-Costantini, R. (2013). Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling. Acta Neuropathologica, 126(4), 575-594. https://doi.org/10.1007/s00401-013-1165-y

Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling. / Cama, Alessandro; Verginelli, Fabio; Lotti, Lavinia Vittoria; Napolitano, Francesco; Morgano, Annalisa; D'Orazio, Andria; Vacca, Michele; Perconti, Silvia; Pepe, Felice; Romani, Federico; Vitullo, Francesca; Di Lella, Filippo; Visone, Rosa; Mannelli, Massimo; Neumann, Hartmut P H; Raiconi, Giancarlo; Paties, Carlo; Moschetta, Antonio; Tagliaferri, Roberto; Veronese, Angelo; Sanna, Mario; Mariani-Costantini, Renato.

In: Acta Neuropathologica, Vol. 126, No. 4, 2013, p. 575-594.

Research output: Contribution to journalArticle

Cama, A, Verginelli, F, Lotti, LV, Napolitano, F, Morgano, A, D'Orazio, A, Vacca, M, Perconti, S, Pepe, F, Romani, F, Vitullo, F, Di Lella, F, Visone, R, Mannelli, M, Neumann, HPH, Raiconi, G, Paties, C, Moschetta, A, Tagliaferri, R, Veronese, A, Sanna, M & Mariani-Costantini, R 2013, 'Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling', Acta Neuropathologica, vol. 126, no. 4, pp. 575-594. https://doi.org/10.1007/s00401-013-1165-y
Cama, Alessandro ; Verginelli, Fabio ; Lotti, Lavinia Vittoria ; Napolitano, Francesco ; Morgano, Annalisa ; D'Orazio, Andria ; Vacca, Michele ; Perconti, Silvia ; Pepe, Felice ; Romani, Federico ; Vitullo, Francesca ; Di Lella, Filippo ; Visone, Rosa ; Mannelli, Massimo ; Neumann, Hartmut P H ; Raiconi, Giancarlo ; Paties, Carlo ; Moschetta, Antonio ; Tagliaferri, Roberto ; Veronese, Angelo ; Sanna, Mario ; Mariani-Costantini, Renato. / Integrative genetic, epigenetic and pathological analysis of paraganglioma reveals complex dysregulation of NOTCH signaling. In: Acta Neuropathologica. 2013 ; Vol. 126, No. 4. pp. 575-594.
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AU - Verginelli, Fabio

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AU - Napolitano, Francesco

AU - Morgano, Annalisa

AU - D'Orazio, Andria

AU - Vacca, Michele

AU - Perconti, Silvia

AU - Pepe, Felice

AU - Romani, Federico

AU - Vitullo, Francesca

AU - Di Lella, Filippo

AU - Visone, Rosa

AU - Mannelli, Massimo

AU - Neumann, Hartmut P H

AU - Raiconi, Giancarlo

AU - Paties, Carlo

AU - Moschetta, Antonio

AU - Tagliaferri, Roberto

AU - Veronese, Angelo

AU - Sanna, Mario

AU - Mariani-Costantini, Renato

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AB - Head and neck paragangliomas, rare neoplasms of the paraganglia composed of nests of neurosecretory and glial cells embedded in vascular stroma, provide a remarkable example of organoid tumor architecture. To identify genes and pathways commonly deregulated in head and neck paraganglioma, we integrated high-density genome-wide copy number variation (CNV) analysis with microRNA and immunomorphological studies. Gene-centric CNV analysis of 24 cases identified a list of 104 genes most significantly targeted by tumor-associated alterations. The NOTCH signaling pathway was the most significantly enriched term in the list (P = 0.002 after Bonferroni or Benjamini correction). Expression of the relevant NOTCH pathway proteins in sustentacular (glial), chief (neuroendocrine) and endothelial cells was confirmed by immunohistochemistry in 47 head and neck paraganglioma cases. There were no relationships between level and pattern of NOTCH1/JAG2 protein expression and germline mutation status in the SDH genes, implicated in paraganglioma predisposition, or the presence/absence of immunostaining for SDHB, a surrogate marker of SDH mutations. Interestingly, NOTCH upregulation was observed also in cases with no evidence of CNVs at NOTCH signaling genes, suggesting altered epigenetic modulation of this pathway. To address this issue we performed microarray-based microRNA expression analyses. Notably 5 microRNAs (miR-200a,b,c and miR-34b,c), including those most downregulated in the tumors, correlated to NOTCH signaling and directly targeted NOTCH1 in in vitro experiments using SH-SY5Y neuroblastoma cells. Furthermore, lentiviral transduction of miR-200s and miR-34s in patient-derived primary tympano-jugular paraganglioma cell cultures was associated with NOTCH1 downregulation and increased levels of markers of cell toxicity and cell death. Taken together, our results provide an integrated view of common molecular alterations associated with head and neck paraganglioma and reveal an essential role of NOTCH pathway deregulation in this tumor type.

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