Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. As microRNAs (miRNAs) are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of miRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified miRNA signatures specific to, as well as common among, the genetic groups of PCCs/PGLs. miRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD-, and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using quantitative real-time PCR. Upregulation of miR-210 in VHL- and SDHB-related PCCs/PGLs was verified, while miR-137 and miR-382 were confirmed as generally upregulated in PCCs/PGLs (except in MAX-related tumors). Also, we confirmed overexpression of miR-133b as VHL-specific miRNAs, miR-488 and miR-885-5p as RET-specific miRNAs, and miR-183 and miR-96 as SDHB-specific miRNAs. To determine the potential roles miRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX tumors allowed us to determine that miRNA regulation occurs primarily through mRNA degradation in PCCs/PGLs, which partially confirmed our miRNA-mRNA integration results.

[1]  S. Sockanathan,et al.  FARP1 Promotes the Dendritic Growth of Spinal Motor Neuron Subtypes through Transmembrane Semaphorin6A and PlexinA4 Signaling , 2009, Neuron.

[2]  Rafael A Irizarry,et al.  Processing of Agilent microRNA array data , 2010, BMC Research Notes.

[3]  W. Nemoto,et al.  ERK5 Activity Is Required for Nerve Growth Factor-induced Neurite Outgrowth and Stabilization of Tyrosine Hydroxylase in PC12 Cells* , 2009, The Journal of Biological Chemistry.

[4]  P. Bondallaz,et al.  Role of the microtubule destabilizing proteins SCG10 and stathmin in neuronal growth. , 2004, Journal of neurobiology.

[5]  P. Munson,et al.  Distinct gene expression profiles in norepinephrine- and epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic pathways in von Hippel-Lindau syndrome. , 2004, Endocrine-related cancer.

[6]  D. Jin,et al.  CREB − a real culprit in oncogenesis , 2007, The FEBS journal.

[7]  J. Sandgren,et al.  MicroRNA profiling of benign and malignant pheochromocytomas identifies novel diagnostic and therapeutic targets. , 2010, Endocrine-related cancer.

[8]  S Miyano,et al.  Open source clustering software. , 2004, Bioinformatics.

[9]  M. Mann,et al.  Universal sample preparation method for proteome analysis , 2009, Nature Methods.

[10]  J. Sznajder,et al.  Elevated CO2 Levels Cause Mitochondrial Dysfunction and Impair Cell Proliferation* , 2011, The Journal of Biological Chemistry.

[11]  C. Marshall,et al.  Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells , 1999, Oncogene.

[12]  J. Song,et al.  MicroRNA‐488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells , 2011, Cell biology international.

[13]  K. Pacak,et al.  First report of bilateral pheochromocytoma in the clinical spectrum of HIF2A-related polycythemia-paraganglioma syndrome. , 2013, The Journal of clinical endocrinology and metabolism.

[14]  W. Kaelin,et al.  Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer. , 2005, Cancer cell.

[15]  T. Furukawa,et al.  MicroRNAs Associated with Mitogen-Activated Protein Kinase in Human Pancreatic Cancer , 2011, Molecular Cancer Research.

[16]  J. Prchal,et al.  A novel EPAS1/HIF2A germline mutation in a congenital polycythemia with paraganglioma , 2012, Journal of Molecular Medicine.

[17]  P Barbry,et al.  miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity , 2011, Cell Death and Differentiation.

[18]  R. Lonser,et al.  Novel HIF2A mutations disrupt oxygen sensing, leading to polycythemia, paragangliomas, and somatostatinomas. , 2013, Blood.

[19]  P. Herrlich,et al.  Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane , 2011, PloS one.

[20]  Patricia L. M. Dahia,et al.  Germline mutations in TMEM127 confer susceptibility to pheochromocytoma , 2010, Nature Genetics.

[21]  Hanah Margalit,et al.  Clustering and conservation patterns of human microRNAs , 2005, Nucleic acids research.

[22]  G. Mortier,et al.  qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data , 2007, Genome Biology.

[23]  Masayuki Kano,et al.  miR‐145, miR‐133a and miR‐133b: Tumor‐suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma , 2010, International journal of cancer.

[24]  G. Stein,et al.  A microRNA signature for a BMP2-induced osteoblast lineage commitment program , 2008, Proceedings of the National Academy of Sciences.

[25]  Y. Akao,et al.  MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation , 2012, Neurochemistry International.

[26]  V. Thayanithy,et al.  Perturbation of 14q32 miRNAs-cMYC gene network in osteosarcoma. , 2012, Bone.

[27]  L. Zhang,et al.  The microRNA expression changes associated with malignancy and SDHB mutation in pheochromocytoma. , 2012, Endocrine-related cancer.

[28]  T. Russo,et al.  miRNA 34a, 100, and 137 modulate differentiation of mouse embryonic stem cells , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[29]  B. Devlin,et al.  Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. , 2000, Science.

[30]  渡邉 可奈子.  Latent process genes for cell differentiation are common decoders of neurite extension length , 2012 .

[31]  E. Kohmura,et al.  MicroRNA-183 upregulates HIF-1α by targeting isocitrate dehydrogenase 2 (IDH2) in glioma cells , 2013, Journal of Neuro-Oncology.

[32]  G. Hannon,et al.  A MicroRNA Feedback Circuit in Midbrain Dopamine Neurons , 2007, Science.

[33]  Zsolt Czimmerer,et al.  DIFFERENTIALLY EXPRESSED MicroRNAs IN SMALL CELL LUNG CANCER , 2009, Experimental lung research.

[34]  J. Castle,et al.  Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs , 2005, Nature.

[35]  J. Loscalzo,et al.  MicroRNA-210: A unique and pleiotropic hypoxamir , 2010, Cell cycle.

[36]  M. Meyerson,et al.  The kinesin KIF1Bbeta acts downstream from EglN3 to induce apoptosis and is a potential 1p36 tumor suppressor. , 2008, Genes & development.

[37]  A. González-Neira,et al.  Genetics of pheochromocytoma and paraganglioma in Spanish patients. , 2009, The Journal of clinical endocrinology and metabolism.

[38]  A. Isaksson,et al.  Stathmin as a marker for malignancy in pheochromocytomas. , 2009, Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association.

[39]  Gabriele Sales,et al.  MAGIA, a web-based tool for miRNA and Genes Integrated Analysis , 2010, Nucleic Acids Res..

[40]  S. Pfaff,et al.  Small CTD Phosphatases Function in Silencing Neuronal Gene Expression , 2005, Science.

[41]  Ramón Díaz-Uriarte,et al.  Pomelo II: finding differentially expressed genes , 2009, Nucleic Acids Res..

[42]  M. Mann,et al.  Quantitative proteomics reveals subset-specific viral recognition in dendritic cells. , 2010, Immunity.

[43]  G. Arnaldi,et al.  Clinically guided genetic screening in a large cohort of italian patients with pheochromocytomas and/or functional or nonfunctional paragangliomas. , 2009, The Journal of clinical endocrinology and metabolism.

[44]  Y. Hasegawa,et al.  ARHGAP18, a GTPase-activating protein for RhoA, controls cell shape, spreading, and motility , 2011, Molecular biology of the cell.

[45]  Agnieszka Maliszewska,et al.  Research resource: Transcriptional profiling reveals different pseudohypoxic signatures in SDHB and VHL-related pheochromocytomas. , 2010, Molecular endocrinology.

[46]  P. Dahia,et al.  Minireview: the busy road to pheochromocytomas and paragangliomas has a new member, TMEM127. , 2011, Endocrinology.

[47]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[48]  C. Cremers,et al.  SDHAF2 mutations in familial and sporadic paraganglioma and phaeochromocytoma. , 2010, The Lancet. Oncology.

[49]  P. Igaz,et al.  MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas , 2010, Modern Pathology.

[50]  Satoru Miyano,et al.  Open source clustering software , 2004 .

[51]  F. Westermann,et al.  MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival , 2011, Cell Death and Differentiation.

[52]  J. Steen,et al.  Pleiotropic effects of miR-183~96~182 converge to regulate cell survival, proliferation and migration in medulloblastoma , 2012, Acta Neuropathologica.

[53]  C. Eng,et al.  The pressure rises: update on the genetics of phaeochromocytoma. , 2002, Human molecular genetics.

[54]  Electron Kebebew,et al.  Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. , 2012, The New England journal of medicine.

[55]  P. Bénit,et al.  The Warburg Effect Is Genetically Determined in Inherited Pheochromocytomas , 2009, PloS one.

[56]  A. Tischler,et al.  New syndrome of paraganglioma and somatostatinoma associated with polycythemia. , 2013, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[57]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[58]  E. Letouzé,et al.  Somatic NF1 inactivation is a frequent event in sporadic pheochromocytoma. , 2012, Human molecular genetics.

[59]  Eunjoon Kim,et al.  Leucine‐rich repeat proteins of synapses , 2007, Journal of neuroscience research.

[60]  P. Bénit,et al.  SDHA is a tumor suppressor gene causing paraganglioma. , 2010, Human molecular genetics.

[61]  S. Srikantan,et al.  In vivo and in vitro oncogenic effects of HIF2A mutations in pheochromocytomas and paragangliomas. , 2013, Endocrine-related cancer.

[62]  Yong Liu,et al.  MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382 , 2010, Nucleic acids research.

[63]  M. Urioste,et al.  MAX Mutations Cause Hereditary and Sporadic Pheochromocytoma and Paraganglioma , 2012, Clinical Cancer Research.

[64]  Xiaorong Li,et al.  miR-133b regulates the MET proto-oncogene and inhibits the growth of colorectal cancer cells in vitro and in vivo , 2010, Cancer biology & therapy.

[65]  G. Pita,et al.  Tumoral EPAS1 (HIF2A) mutations explain sporadic pheochromocytoma and paraganglioma in the absence of erythrocytosis. , 2013, Human molecular genetics.

[66]  G. Shadel,et al.  Revisiting the TCA cycle: signaling to tumor formation. , 2011, Trends in molecular medicine.

[67]  Yi Zhao,et al.  Clustered microRNAs' coordination in regulating protein-protein interaction network , 2009, BMC Systems Biology.

[68]  P. M. Dahia,et al.  Transcription Association of VHL and SDH Mutations Link Hypoxia and Oxidoreductase Signals in Pheochromocytomas , 2006, Annals of the New York Academy of Sciences.

[69]  J. Benítez,et al.  Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma , 2011, Nature Genetics.

[70]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[71]  A. Ferguson-Smith,et al.  Epigenetic alteration at the DLK1-GTL2 imprinted domain in human neoplasia: analysis of neuroblastoma, phaeochromocytoma and Wilms' tumour , 2005, British Journal of Cancer.

[72]  G. Riggins,et al.  Altered cancer cell metabolism in gliomas with mutant IDH1 or IDH2 , 2012, Current opinion in oncology.