Open Research Online The Open University’s repository of research publications and other research outputs Role of genes differentially expressed in thyroid carcinogenesis Thesis How to cite: Anania, Maria Chiara (2012). Role of genes differentially expressed in thyroid carcinogenesis. PhD thesis The Open University. For guidance on citations see FAQs. c 2012 The Author https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000eeae Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk o FONDAZIONE IRees +-it.•. lsmuro NAZIONALE l±lT DEI TUMOI.I Maria Chiara Anania Degree in Biological Science OU personal identifier A298071X ROLE OF GENES DIFFERENTIALLY EXPRESSED IN THYROID CARCINOGENESIS This thesis is presented to The Open University for the Degree of Doctor of Philosophy Discipline: Life and Biomolecular Sciences Date of submission: 31st May 2012 Affiliated Research Centre: Fondazione IRCCS Istituto Nazionale dei Tumori, Milan (Italy) Director of studies: Dr. Angela Greco External supervisor: Dr. Karen Pulford 1 IMAGING SERVICES NORTH Boston Spa, Wetherby West Yorkshire, LS23 7BQ www.bl,uk BEST COpy AVAILABLE. VARIABLE PRINT QUALITY NOTIFICATION OF REDACTION THESIS TITLE: Role of genes differentially expressed in thyroid carcinogenesis. AUTHOR: Maria Chiara Anania YEAR: 2012 CLASSMARK: 616.99444 ANA The following pages/sections have been redacted from this thesis: Paqe No. Item/section redacted p.l1~ - End PUBLICATIONS ETHOS/SUREDACTION NOTIFICATION 03/2016_1 TABLE OF CONTENTS ABSTRACT 5 1. INTRODUCTION 8 1.1 Thyroid cancer 9 1.2 Histological classification of follicular cell-derived thyroid tumors 11 1.2.1 Papillary thyroid carcinoma (PTC) 11 1.2.2 Follicular thyroid carcinoma (FTC) 12 1.2.3 Poorly Differentiated thyroid carcinoma (PDTC) 13 1.2.4 Anaplastic thyroid carcinoma (ATC) 13 1.3 Genetic alteration in thyroid cancer 14 1.3.1 v-Rafmurine sarcoma viral oncogene homolog Bl (BRAF) oncogenes 14 1.3.2 REarranged during Transfection (RET) oncogenes 16 1.3.3 TRK oncogenes 18 1.3.4 RAS mutation 20 1.3.5 Paired box gene 8IPeroxisome proliferator-activate receptor (P AX8IPP AR'Y) rearrangement 21 1.3.6 PI3KJAKT pathway mutations 22 1.3.7 Tumor protein 53 (TP53) mutation 23 1.3.8 Other alterations in thyroid cancer 24 1.4 Meccanisms of thyroid cancer aetiology 30 1.5 Therapy in thyroid cancer 33 1.5.1 Multikinase inhibitors as target therapy 33 1.5.2 Conclusions on therapeutic options 35 2. EXPRESSION PROFILES IN THYROID CANCER 37 2.1 Expression profiles in thyroid cancer 38 2.1.1 PTC signature 38 2.1.2 Post Chemobyl-PTC signature 41 2.1.3 FTC signature 42 2.1.4 PDTC and ATC signature 43 2.2 miRNA profiling in thyroid cancer 45 2.3 Gene silencing by promoter methylation in thyroid cancer 46 2.4 Conclusions of gene expression studies 50 AIM OF THE THESIS 51 2 3. MATERIALS AND METHODS 52 3.1 Microarray data sets and statistical analysis 53 3.2 Molecular biology 55 3.2.1 RNA extraction and RT-PCR 55 3.2.2 Real-time RT-PCR 55 3.2.3 Construction of expression vectors 55 3.3 Cell biology 58 3.3.1 Cell lines 58 3.3.2 Cell treatments 59 3.3.3 Cell transfection 60 3.3.4 TIMP 3 and S1 OOA 11 silencing 60 3.3.5 Focus forming assay 61 3.3.6 Cell cycle analysis 61 3.3.7 Growth curves 62 3.3.8 Colony forming assay 62 3.3.9 Cell adhesion assay 62 3.3.10 Wound healing assay 63 3.3.11 Migration and invasion assay 63 3.3.12 Soft agar assay 64 3.4 Biochemical assay and studies 64 3.4.1 Western blot analysis 64 3.4.2 Dot blot analysis 65 3.4.3 TNF-a detection 66 3.5 In vivo studies 66 3.5.1 Immunohistological studies 66 4. TIMP3 STUDIES 68 4.1 Introduction 69 4.1.1 TIMP3 69 4.1.2 TIMP3 and thyroid cancer 72 4.2 Aims of the chapter 73 4.3 Results 74 4.3.1 Expression analysis of TIMP3 in PTC samples and thyroid tumour celllines 74 4.3.2 Effect ofTIMP3 restoration on cell growth 76 4.3.3 Effect of TIMP3 restoration on cell adhesion 81 4.3.4 Effect of TIMP3 restoration on cell migration and invasion 83 3 4.3.5 Effect ofTIMP3 restoration on anchorage independent growth 85 4.3.6 Analysis of proteins mediating TIMP3 effects 86 4.3.7 Effect ofTIMP3 restoration in mouse tumour xenografts 88 4.4 Discussion 92 5. SlOOAll STUDIES 96 5.1. Introduction 97 5.1.1 SIDOAII 97 5.1.2 SIOOAII and thyroid cancer ID4 5.2 Aims of the chapter 105 5.3 Results 106 5.3.1 Expression analysis of S 1OOA 11 in PTC samples and PTC derived cell lines 106 5.3.2 Analysis of cellular localization of S I OOA 11 109 5.3.3 Analysis of interat ion between SIOOAII and EGF/EGFR pathway 112 5.3.4 Effect ofSIOOAll silencing 114 5.3.4.1 Transient silencing ofS100All 114 5.3.4.2 Stable silencing ofSIOOAII 116 5.3.4.3 Effect of stable SIDOAll silencing in mouse tumour xenografts 120 5.3.5 Effect ofSIOOAll on the transforming potential of TRK-T3 oncogene 122 5.3.5.1 SIOOAII enhances "in vitro" TRK-T3 trasforming activity 122 5.3.5.2 Biochemical and biological analysis ofT3/SI00 foci 123 5.3.5.3 Analysis of the tumorigenic capability ofT3/S100 foci 127 5.4 Discussion 129 6. CITED 1 STUDIES 134 6.1 Introduction 135 6.2 Preliminary results 136 7. IGFBP7 STUDIES 139 8. GENERAL DISCUSSION AND FUTURE PLANS 142 REFERENCES 148 LIST OF ABBREVIATIONS 174 LIST OF FIGURES 175 LIST OF TABLES 176 PUBLICATIONS 177 ACKNOWLEDGEMENTS 178 4 Abstract Thyroid cancer represents the most common endocrine malignancy, and its incidence has increased significantly over the last few decades. Papillary thyroid carcinoma (PTC), the most frequent neoplasia originating from the thyroid epithelium, accounts for about 80% of aU thyroid cancers. PTC is characterized by rearrangements of RET and NTRKl receptor tyrosine kinases, or by activating point mutations in the BRAF serine/threonine kinase or in the RAS genes. Even though the identification of PTC-associated oncogenes has provided a great contribution to the understanding of PTC pathogenesis, the molecular mechanisms underlying the development of this neoplasia, including the role of tumour suppressor genes, are still far from being completely elucidated. Recently, global gene expression analyses have provided new findings contributing to the dissection of thyroid tumour pathogenesis, through the identification of genes discriminating among different histotypes, candidates as new therapeutic targets and possible tumour suppressor genes. Despite the numerous gene expression studies, there are few data addressing the role of differentially expressed genes in the pathogenesis of thyroid tumours. A microarray gene expression profile previ~usly determined in our laboratory identified a list of genes differentially expressed in PTC versus normal thyroid; among those, we selected TIMP3, SlOOAll and CITEDl genes for which a role in the pathogenesis of PTC was also suggested by recently published gene and protein expression data. In this PhD project, we performed functional studies in order to assess the role of TIMP3, SlOOAll and CITEDl genes in thyroid carcinogenesis, with the aim of unveiling novel mechanisms and identifying novel therapeutic targets in thyroid tumours. TIMP3 (Tissue Inhibitor of Metalloproteinases-3) is a secreted protein able to inhibit extracellular matrix metalloproteinases. TIMP3 gene promoter has been found hypermethylated in thyroid cancer and its downregulation was associated with several aggressive tumour features. To investigate the role of TIMP3 in the pathogenesis of PTC 5 we used an integrated approach including analysis of several gene expression data sets and functional studies. TIMP3 was found to be downregulated in a consistent fraction in PTCs, with respect to normal thyroid. Restoration of TIMP3 in the PTC-derived NIMI cell line had no effect on growth rate; however, it reduced migration, invasion and anchorage independent growth. The striking effect was observed in vivo, as TIMP3 reduced the tumourigenicity ofNIMI cells by repressing angiogenesis and macrophage infiltration. All these observations suggest a tumour suppressor role in thyroid carcinogenesis. S100All (calgizzarin) is a member of the S100 Ca2+-binding protein family, which includes at least 20 proteins. Its role in tumour is not well known, and it appears to have distinct functions in different tumour types. Our microarray analysis showed that S I OOA 11 is overexpressed in PTC compared to normal thyroid. In order to study the role of S 1OOA II in thyroid carcinogenesis, we performed functional in vitro and in vivo analysis. Analysis of cellular localization in PTC-derived K 1 cell line, revealed that S I OOA 11 was mainly cytoplasmic and was able to trans locate into the nucleus after Ca2+and TGF-p stimulation. We also found that this translocation did not increase p21 level, a negative regulator of cell growth. Moreover, we found that S 100A 11 did not alter the activation level of the EGFIEGFR pathway. We then investigated the effect ofS100All silencing on tumourigenic properties of K 1 cells. We found that S 100A 11 silencing did not affect cell proliferation, but it exerted a role on the anchorage-independent growth.