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University of Napoli Federico Ii UNIVERSITY OF NAPOLI FEDERICO II Doctorate School in Molecular Medicine Doctorate Program in Genetics and Molecular Medicine Coordinator: Prof. Lucio Nitsch XXVII Cycle “Characterization of Twist1 transcriptional targets in thyroid cancer cells” Francesca Maria Orlandella Napoli 2015 “Characterization of Twist1 transcriptional targets in thyroid cancer cells” TABLE OF CONTENTS LIST OF PUBLICATIONS 4 ABBREVIATIONS 5 ABSTRACT 7 1. BACKGROUND 8 1.1 Thyroid tumors 8 1.1.1 Morphological and clinical features of follicular cell derived thyroid tumors 8 1.1.2 Genetic alterations in follicular cell derived thyroid tumors 11 1.1.3 Aberrant expression of miRNAs in thyroid cancers 17 1.1.4 Targeted therapy for thyroid carcinomas 21 1.2 The transcription factor Twist1 23 1.2.1 Molecular structures of the Twist1 gene and protein 23 1.2.2 Physiological functions of Twist1 protein 27 1.2.3 Twist1 expression in cancers 28 1.2.4 Mechanisms of Twist1 reactivation in human cancers 29 1.3.5 Downstream effects of Twist1 in human cancers 31 2. AIMS OF THE STUDY 35 3. MATERIALS AND METHODS 36 3.1 Reagents 36 3.2 Cell lines 36 3.3 Tissue samples 36 3.4 Immunohistochemistry 37 3.5 Microarray analysis 37 3.6 Network and gene ontology analysis 37 3.7 RNA extraction, cDNA synthesis, and quantitative real-time PCR 38 3.8 RNA silencing 38 3.9 Cell Proliferation Assays 38 3.10 Transwell and Collagen cell Migration Assay, Collagen Cell Invasion Assay, Transendothelial Cell Migration Assay 39 3.11 Chromatin immunoprecipitation assay 39 3.12 miR-584 transfection 39 3.13 Wound Closure and Matrigel Invasion Assay 39 3.14 Protein studies 40 3.15 Luciferase Assays 40 3.16 Statistical analysis 40 4. RESULTS 41 4.1 Identification of mRNA targets of Twist1 41 4.1.1 mRNA expression profile of TPC-Twist1 cells 41 4.1.2 Silencing of Twist1 up-regulated genes in TPC-Twist1 and in CAL62 cells impairs cell viability 44 4.1.3 Silencing of HS6ST2, THRB, ID4, RHOB, and PDZK1IP1 in TPC-Twist1 and in CAL62 cells impairs cell migration and invasion 45 2 4.1.4 Twist1 directly binds the promoter of target genes 49 4.1.5 Twist1 target genes are over-expressed in PTC, PDC, and ATC samples 50 4.2 Identification of Twist1 target miRNAs 52 4.2.1 miRNome profiles of TPC-Twist1 cells 52 4.2.2 miR-584 is up-regulated in ATC samples 54 4.2.3 Ectopic expression of miR-584 protects TPC cells from apoptosis 55 4.2.4 Silencing of miR-584 in TPC-Twist1 cells induces apoptosis 58 4.2.5 miR-584 targets TUSC2 61 4.2.6 TUSC2 is down-regulated in human thyroid tumors 64 5. DISCUSSION 66 6. CONCLUSIONS 72 7. ACKNOWLEDGEMENTS 73 8. REFERENCES 74 3 LIST OF PUBLICATIONS This dissertation is based upon the following publications: I. Di Maro G, Orlandella FM, Bencivenga TC, Salerno P, Ugolini C, Basolo F, Maestro R, Salvatore G. Identification of targets of Twist1 transcription factor in thyroid cancer cells. J Clin Endocrinol Metab. 2014 Sep;99(9):E1617-26. doi: 10.1210/jc.2013-3799. Epub 2014 May 21. PubMed PMID: 24848707. II. Di Maro G, Salerno P, Unger K, Orlandella FM, Monaco M, Chiappetta G, Thomas G, Oczko-Wojciechowska M, Masullo M, Jarzab B, Santoro M, Salvatore G. Anterior gradient protein 2 promotes survival, migration and invasion of papillary thyroid carcinoma cells. Mol Cancer. 2014 Jun 30;13:160. doi: 10.1186/1476-4598-13-160. PubMed PMID: 24976026; PubMed Central PMCID: PMC4094684. III. Orlandella FM et al. Twist1-miR-584-TUSC2 pathway protects thyroid cancer cells from apoptosis. (Manuscript in preparation). 4 ABBREVIATIONS AKT v-akt murine thymoma viral oncogene ALK Anaplastic lymphoma kinase ATC Anaplastic thyroid carcinoma bHLH Basic helix-loop-helix BRAF B-type RAF CBP CREB-binding protein CCDC6 Coiled-coil domain containing gene 6 CREB cAMP-response element binding protein CTNNB1 Catenin Beta 1 (cadherin-associated protein) DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide dNTPs Deoxyribonucleoside triphosphates ECM Extracellular matrix EGFR Epidermal growth factor receptor EMT Epithelial mesenchymal transition FBS Fetal bovine serum FDA Food and drug administration FNA Fine needle aspiration FTC Follicular thyroid carcinoma GDNF Glial-derived neurotrophic factor GFRα GDNF family receptor alpha HDAC Histone deacetylases IC50 50% growth-inhibitory concentration IDH1 Isocitrate dehydrogenase 1 mAb Monoclonal antibodies MAPK Mitogen-activated protein kinase miRNA MicroRNA mRNA RNA messenger NcoA4 Nuclear receptor co-activator gene 4 NF-Kb Nuclear factor-kappaB NLS Nuclear localization signal NT Normal thyroid NTRK1 Neurotrophic tyrosine kinase receptor type 1 PCAF P300/CBP associated factor PDC Poorly differentiated carcinoma PI3KCA Phosphatidylinositol-3 kinase catalytic domain PPARγ Peroxisome proliferation activated receptor PTC Papillary thyroid carcinoma PTEN Phosphatase and tensin homolog RAI Radioactive iodine therapy RET Rearranged during transfection RTK Tyrosine kinase receptors 5 SCS Saethre-Chotzen syndrome siRNA Small interference RNA TAM Tumor associated macrophages TCGA The Cancer Genome Atlas TERT Telomerase Reverse Transcriptase TSP-1 Thrombospondin-1 UTR Untranslated regions WHO World Health Organization WT Wild type 6 ABSTRACT Follicular cell derived thyroid carcinoma comprise a heterogeneous group of cancers with different clinical and morphological characteristics. Anaplastic thyroid carcinomas (ATC) represent less than 5% of all thyroid cancers but are responsible for more than 50% of thyroid cancer mortality, with a mean survival time from diagnosis of ∼3–6 months. ATC is characterized by local invasion, distant metastasis, chemo and radio-resistance. We showed that ~ 50% of ATCs up-regulated Twist1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Twist1 is a highly conserved basic helix-loop-helix transcription factor that plays an important role in the development and progression of human cancer. Knockdown of Twist1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of Twist1 in papillary thyroid carcinoma cells (TPC) induced resistance to apoptosis and increased cell migration and invasion. In this dissertation, we have investigated the molecular mechanisms by which Twist1 exerts its biological effect in thyroid cancer cells. To this aim, we analyzed gene expression profiles of thyroid cancer cells ectopically expressing Twist1 in comparison to vector control cells. According to gene expression profile, the top functions enriched in TPC-Twist1 cells were: cellular movement, cellular growth and proliferation, cell death and survival. Silencing of the top ten up-regulated genes reduced viability of TPC-Twist1 and CAL62 cells. Silencing of COL1A1, KRT7, PDZK1 induced also cell death. Silencing of HS6ST2, THRB, ID4, RHOB, and PDZK1IP, also impaired migration and invasion of TPC-Twist1 cells. Chromatin immunoprecipitation showed that Twist1 directly binds the promoter of the ten up-regulated genes. q-RT-PCR on thyroid cancer samples showed that HS6ST2, COL1A1, F2RL1, LEPREL1, PDZK1 and PDZK1IP1 are over-expressed in thyroid carcinoma samples compared to normal thyroids. More recently we have conducted a miRNome expression profile of TPC-Twist1 cells in comparison to control. We identified a set of miRNA potentially regulated by Twist1. We then focused on miR-584 up-regulated by Twist1 in TPC cells. We showed that miR-584 is up-regulated in ATCs respect to normal thyroids and papillary thyroid carcinomas. The ectopic expression of miR-584 protected TPC cells from apoptosis induced by Doxorubicin and Staurosporine. Finally, we showed that miR-584 targets TUSC2 (tumor suppressor candidate 2). Thus, we have identified a set of mRNA and miRNA that mediates Twist1 biological effects in thyroid cancer cells. Transcription factors as Twist1 are generally believed to be difficult to target due to nuclear localization and lack of a specific groove for tight binding of a small molecule inhibitor. Thus, downstream targets of Twist1 could be more realistic for therapeutic intervention. 7 1. BACKGROUND 1.1 Thyroid tumors 1.1.1 Morphological and clinical features of follicular cell derived thyroid tumors Thyroid cancer is a common type of endocrine malignancy and its incidence has been steadily increasing worldwide (Jung et al. 2014). The enhanced incidence is limited to the papillary type of thyroid cancer (Pellegriti et al. 2013) and it is believed to result from increased access to high-resolution imaging (particularly ultrasonography) and increased use of fine needle aspiration (FNA) biopsy of small nodules, as well as progressively decreasing stringency of histopathologic criteria applied to the diagnosis of papillary cancer during the past 10-15 years (Howlader et al. 2012). There are several histological types and subtypes of thyroid cancer with different cellular origins, characteristics and prognoses. The vast majority of thyroid tumors arise from thyroid follicular epithelial cells, whereas ~3-5% of cancers originate from parafollicular or C cells (Xing 2013). The follicular cell-derived cancers (~95% of cases) are further subdivided into well- differentiated papillary (PTC) carcinoma and follicular carcinoma (FTC), poorly differentiated carcinoma (PDC) and anaplastic (undifferentiated) carcinoma (ATC). Less-differentiated thyroid cancers (PDC and ATC) can develop de novo or through a stepwise dedifferentiation of papillary and follicular carcinomas (Figure 1) (Pitoia et al. 2013). 8 Figure 1. Model of thyroid multi-step carcinogenesis. Scheme of step-wise dedifferentiation of follicular cell-derived thyroid carcinoma with underlined main histological features. PTCs comprise ~75-85% of all thyroid cancers and represent the most common pediatric thyroid malignancy. Most PTCs in adults occur in patients between 20 and 50 year of age and it is more frequent in women than men (Nikiforov and Nikiforova 2011). Multifocality, lymphatic or local spreading and lymph node metastases are frequent features of PTC whereas distant metastases are relatively uncommon (Omur and Baran 2014). The overall 5 and 10-year survival rates of PTCs are ~98% and 95%, respectively (Jemal et al.
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