Universidad de Oviedo Programa de Doctorado “Investigación en Cáncer” “MOLECULAR MECHANISMS INVOLVED IN THE PATHOGENESIS OF HEAD AND NECK PARAGANGLIOMAS” TESIS DOCTORAL ANNA MERLO 23/06/2014 Universidad de Oviedo Programa de Doctorado “Investigación en Cáncer” “MOLECULAR MECHANISMS INVOLVED IN THE PATHOGENESIS OF HEAD AND NECK PARAGANGLIOMAS” TESIS DOCTORAL ANNA MERLO 23/06/2014 INDEX 1. INTRODUCTION…………………………………………………………………………………….2 1.1 The neural crest………………………………………………………………………………...2 1.2 The paraganglion system………………………………………………………………………3 1.2.1 The carotid body……………………………………………………………………….5 1.2.2 Adrenal medulla………………………………………………………………………..7 1.3 Paraganglioma and pheocromocitomas………………………………………………………7 1.4 Genetic background of HNPGLs and PGL/PCC…………………………………………….8 1.4.1 Von Hippel Lindau disease…………………………………………………………...9 1.4.2 Neurofibromatosis Type 1…………………………………………………………...10 1.4.3 Multiple Endocrine Neoplasia Type 2………………………………………………10 1.4.4 SDH mutation…………………………………………………………………………10 1.4.5 Trasmembran protein 127 (TMEM127)……………………………………………13 1.4.6 1.4.6. Myc-associated factor X (MAX)……………………………………………..13 1.4.7 Hypoxia inducible factor 2 alpha (HIF-2α)………………………………………....13 1.4.8 1.4.8. Fumarate Hydratase (FH)……………………………………………………14 1.4.9 1.4.9. Prolyl hydroxylase domain 2 and Kinesin family member 1B…………..14 1.5 Genetic testing…………………………………………………………………………………14 1.6 Hypoxia and tumorogenesis………………………………………………………………….15 2. OBJECTIVES……………………………………………………………………………………….18 3. MATERIALS AND METHODS…………………………………………………………………....19 3.1 Biological sample……………………………………………………………………………....20 3.2 Tumors and normal paraganglia samples…………………………………………………..20 3.3 Cell culture……………………………………………………………………………………...22 3.4 Chemical treatments…………………………………………………………………………..23 3.4.1 DMOG treatment………………………………………………………………………..23 3.4.2 TTFA treatment………………………………………………………………………….23 3.5 SiRNA treatment and transient transfection…………………………………………………24 3.6 In situ mutagenesis…………………………………………………………………………….25 3.7 Plasmid isolation……………………………………………………………………………….27 3.8 Bacterial culture medium…………………………………………………………………......27 3.9 DNA extraction…………………………………………………………………………………28 3.10 RNA extraction………………………………………………………………………………..29 3.11 cDNA synthesis……………………………………………………………………………….30 3.12 Real Time q-PCR with Syber Green® Master Mix (Life Technologies)………………...30 3.13 cDNA synthesis “RT-PCR TaqMan MicroRNA Assay Kit” (Applied Biosystem)………31 3.14 Real Time q-PCR with TaqMan 2X Universal PCR Master Mix…………………………32 3.15 Multiplex Ligation Probe-dependent Amplification………………………………………..32 3.16 Real time q-PCR method to confirm results from MLPA…………………………………35 3.17 Western blot…………………………………………………………………………………..36 3.18 Bisulfite Conversion and Pyrosequencing…………………………………………………36 3.18.1 Bisulfite Conversion…………………………………………………………………...37 3.18.2 Bisulfite Pyrosequenciation…………………………………………………………..37 3.19 Mutation analysis………………………………………………………………………………38 3.20 Polymerase Chain reaction (PCR)…………………………………………………………..38 3.21 Purification of PCR products…………………………………………………………………42 3.22 Immunocitochemestry………………………………………………………………………...42 3.23 Inmunofluorimetry …………………………………………………………………………….43 3.24 Immunohistochemestry……………………………………………………………………….43 3.25 Microarrays and TaqMan Low-Density Array (TLDA)……………………………………..44 3.26 Vascular Density………………………………………………………………………………44 3.27 Statistical Analysis…………………………………………………………………………….45 3.28 Microarray data analysis……………………………………………………………………...45 3.29 Bioinformatics Analysis of Oncomine Cancer Gene Microarray Database……………..45 4. RESULTS…………………………………………………………………………………………...47 4.1 Expression profiles analysis of HNPGLs vs normal samples……………………………..48 4.2 Validation of microarrays data………………………………………………………………..56 4.3 Relationship between the pseudohypoxic gene signatures of HNPGLs and the SDH mutation status……………………………………………………………………………………..57 4.4 C1-tumor analysis for somatic mutation in SDH……………………………………………58 4.5 Vascular density of C1-C2-HNPGLs and normal paraganglia……………………………58 4.6 Analysis of HIF-1α and HIF-2 α protein expression in HNPGL…………………………...59 4.7 Hypoxia related miR-210 is overexpressed in C1-HNPGL………………………………..60 4.8 Link between hypoxia/HIF-1α and ISCU and miR-210 expression………………………62 4.9 Identification of negative SDHB immunostaining in association with deregulation of HIF- 1α, miR-210, and ISCU……………………………………………………………………………65 4.10 Validation of the data on HIF-1α/miR-210/ISCU/SDHB pathway in HNPGLs…………68 4.11 Mecanisms of HIF-1α upregulation in non-SDHx HNPGLs………………………….......70 4.12 Somatic VHL mutations in HNPGLs in association with HIF-1α/miR-210 gene signature…………………………………………………………………………………………….72 4.13 Oncomine meta-analysis reveals similarities of pHx-HNPGL with ccRCC…………….77 4.14 VHL muation is involved in the activacion of the hypoxia signaling via HIF-1α……….80 4.15 pVHL loss of expression and VHL deletion do not activate HIF-1α/CAIX pathway in HNPGL………………………………………………………………………………………………81 4.16 HIF-1α is required for miR-210 over-expression…………………………………………82 4.17 VHL mutation F76del activates HIF-1α/CaIX/miR-210 pathway……………………….83 4.18 Loss of SDH does not activate the hypoxic pathway…………………………………….86 4.19 Analysis of VHL gene alteration in HNPGLs………………………………………………87 4.20 miRNA expression profiling of HNPGLs…………………………………………………...93 4.21 PGL2, PGL3 and PGL7 differ from the rest of HNPGLs in the expression of genes involved in differentiation and energetic metabolism…………………………………………..96 4.22 “Cluster reverse”……………………………………………………………………………...98 5. DISCUSSION……………………………………………………………………………………….100 5.1 Sporadic and SDH- HNPGLs show a common gene expression signature…………..101 5.2 Pseudohypoxic gene signature is driven by HIF-1α activation in a subgroup of HNPGLs…………………………………………………………………………………………..101 5.3 A robust and distinct pseudohypoxic HIF-1α-related miRNA profile defines a subset of sporadic HNPGLs………………………………………………………………………………..102 5.4 Comparison of the pseudohypoxia/hypoxia transcriptome common to all parasympathetic HNPGLs with that of sympathetic PGLs…………………………………..107 5.5 C1-HNPGLs share a common hypoxic profile with ccRCC……………………………..109 5.6 Identification of somatic VHL genes mutation in sporadic HNPGLs with psuedohypoxic gene expression profile………………………………………………………………………….110 5.7 Somatic VHL deletions are common in sporadic HNPGLs……………………………..113 5.8 VHL mutation p.F76del led to the activation of HIF1-α/CAIX/miR-210 pathway……..115 5.9 MAX muation analysis in HNPGLs………………………………………………………...115 5.10 miRNA expression profiling of HNPGLs…………………………………………………115 5.11 Part of the cluster mir-379/mir-656 is found downregulated in PGL2, PGL3 and PGL7………………………………………………………………………………………………116 5.12 miRNAs founded down-regulated in all NPGLs but not in PGL2, PGL3 and PGL7..119 6. CONCLUSIONES…………………………………………………………………………………..118 7. BIBLIOGRAPHY……………………………………………………………………………………120 8. SUPPLEMENTAL MATERIAL…………………………………………………………………….141 1. INTRODUCTION 1. INTRODUCTION Paragangliomas and pheocromocytomas are rare tumors arising from parasympathetic and sympathetic paraganglias through the body. Paraganglias are part of the autonomic nervous system that derives from embryonic neural crest. 1.1 The neural crest. The neural crest is an embryonic stem cell population unique to vertebrate that contribute to a wide variety of cells derivatives like sensory and autonomic ganglia of peripheral nervous system, adrenomedullary cells, cartilage and bone of the face, and pigmentation of the skin (1). During its formation, in the embryonic development, neural crest undergoes an extensive morphogenetic movement (Fig.1). Initially the cells that will form neural crest are located in the neural plate border, at the edges of the neural plate which is the embryonic region that will form the central nervous system. During the neurolation, the neural plate invaginates by elevation of the edge forming a cylindrical structure called neural tube which will later form the brain and spinal cord. The premigratory neural crest cells, during the process of neural tube closure, converge toward the midline and are located in the dorsal part of the neural tube. Then, they lose the intercellular connection and undergo an epithelial to mesenchymal transition (EMT), and acquire the ability to migrate and leave the neural tube (2, 3). 2 Figure 1. Neural crest formation. (A) Schematic diagram of transverse sections through embryo during neurulation. Cells that will form neural crest are located in the neural plate border (green), a territory between the neural plate and the non-neural ectoderm. (B) The neural plate invaginates, resulting in the elevation of the neural folds, which contain neural crest precursors. (C) After neural tube closure, neural crest cells lose intercellular connections and undergo an epithelial to mesenchymal transition and adcquire the capacity of migrate. (Image taken from reference 1). Neural crest cells emigrate and populate different niches throughout the embryo giving rise to different cell types and contribute to the formation of a variety of tissues and organs. 1.2 The paraganglion system. Some of the neural crest cells migrate beyond the pre-vertebral and para-vertebral parasympathetic chains or beyond the sympathetic chains and take on glandular character resulting in the paraganglion system. Paraganlia are dispersedly diffuse from 3 the middle ear and the skull base to the pelvic floor and are classified as either sympathetic or parasypathetic (4). Sympathetic paraganglia are located along the prevertebral and paravertebral sympathetic