SYNDECAN-1 INSIGHTS IN MESOTHELIOMA THESIS FOR DOCTORAL DEGREE ( Ph . D . ) By Ghazal

Syndecan-1 is a cell surface heparan sulfate proteoglycan expressing on epithelial cells. Heparan sulfate (HS) chains on syndecan-1 constitute growth factor attachment sites and facilitate growth factors to bind their respective receptors. This binding property of HS chains allows syndecan-1 to be involved in various cellular processes. The fine structure of HS defines the binding properties of these chains. Sulfatase-1 is one of the enzymes that regulates sulfation pattern of HS chains. On the cell membrane syndecan-1 can get shed and the soluble proteoglycan can compete with cell surface-bound and might have counteracting roles. Malignant mesothelioma (MM) is highly aggressive tumor of mesothelial cells lining the serosal cavities. Presence of syndecan-1 on the cell surface of MM is associated with favorable prognosis, whereas the decrease of syndecan-1 deteriorates the prognosis. With this thesis work, we aimed to disclose syndecan-1 roles and the underlying mechanisms by which syndecan-1 affect the behavior of malignant mesothelioma. We focused on genes and pathways modulated by syndecan-1 overexpression and silencing in a mesothelioma cell line (paper I), and we found out that TGF-β, EGF, VEGF and ERK/MAPK pathways were affected in both settings. Syndecan-1 silencing enriched cell cycle pathways and syndecan-1 overexpression had the opposite effects. Syndecan-1 overexpression affected gene expression involved in angiogenesis, adhesion, proliferation, cell cycle, migration, interleukins, extracellular matrix proteins and HS modifying enzymes. Among the HS modifying enzymes affected by syndecan-1 overexpression, sulfatase-1 gene was highly downregulated (paper III). HS content was decreased but overall sulfation was increased by syndecan-1 overexpression. Studying downstream signaling molecules showed that syndecan-1 affects PI3K and MAPK signaling pathways in mesothelioma, which leads to cell cycle arrest at G1. Syndecan-1 level was evaluated in two cohorts of patients both in pleural effusions and sera (paper II). Syndecan-1 was elevated in malignant effusions than benign conditions and could predict malignant disease. In addition, patients with higher levels of syndecan-1 in pleural effusion had shorter survival compared to the patients with lower syndecan-1 levels. However these effects were not observed with syndecan-1 levels in serum. Syndecan-1 overexpression on mesothelioma cells inhibited endothelial cell proliferation, migration and tube formation (paper IV). Endothelial cell tube formation was reverted by MMP7 silencing, which is one of the important sheddases of syndecan-1. Co-cultured HUVEC and mesothelioma cells showed less nuclear Yes-associated protein (YAP) expression, which is associated with less migration. The angiogenesis inhibitory effects of syndecan-1 overexpressing cells were conducted by both proand anti-angiogenic factors comprising Angiopoietin-1, FGF-4, HGF, NRG1-β1, TSP-1, TIMP-1 and TGF-β1. VEGF levels in pleural effusions from mesothelioma patients correlate to soluble syndecan-1 levels and have prognostic value in these patients. Combining shed syndecan-1 and VEGF seemed to be better for prognostic evaluation of mesothelioma patients than these factors alone. LIST OF SCIENTIFIC PAPERS I. Szatmári T, Mundt F, Heidari-Hamedani G, Zong F, Ferolla E, Alexeyenko A, Hjerpe A, Dobra K. Novel genes and pathways modulated by syndecan-1: implications for the proliferation and cell-cycle regulation of malignant mesothelioma cells. PLoS One. 2012;7(10):e48091. II. Mundt F, Heidari-Hamedani G, Nilsonne G, Metintas M, Hjerpe A, Dobra K. Diagnostic and prognostic value of soluble syndecan-1 in pleural malignancies. Biomed Res Int. 2014;2014:419853. III. Heidari-Hamedani G, Vivès RR, Seffouh A, Afratis NA, Oosterhof A, van Kuppevelt TH, Karamanos NK, Metintas M, Hjerpe A, Dobra K, Szatmári T. Syndecan-1 alters heparan sulfate composition and signaling pathways in malignant mesothelioma. Cell Signal. 2015 Oct;27(10):2054-67. IV. Heidari-Hamedani G, Javadi J, Schmalzl A, Szatmári T, Metintas M, Aspenström P, Hjerpe A and Dobra K. Syndecan-1 overexpressing mesothelioma cells inhibit proliferation, migration and tube formation of endothelial cells by synchronized action of angiogenesis related factors. Manuscript. TABLE OF CONTENTS 1 Background . 7 1.1 Malignant mesothelioma . 7 1.1.1 Types, aetiology and epidemiology . 7 1.1.2 Pleural effusions . 7 1.1. 3 Diagnosis of malignant pleural mesothelioma . 8 1.1. 4 Molecular pathogenesis of malignant mesothelioma . 11 1.1. 5 Role of intracellular signaling in malignant pleural mesothelioma . 12 1.1. 6 Angiogenesis in malignant pleural mesothelioma . 13 1.1. 7 Treatment of malignant mesothelioma . 14 1.2 Syndecan-1 biology . 15 1.2. 1 Proteoglycans . 15 1.2. 2 Syndecans . 15 1.2. 3 Syndecans’ structure . 16 1.2. 4 Ectodomain shedding of syndecans . 18 1.2. 5 Syndecan-1, prototype member of the syndecan family . 19 1.2. 6 Syndecan-1 in cancer . 20 2 Aims . 25 3 Remarks on methodology . 26 3.1 Cell lines . 26 3.2 Patient materials . 26 3.3 Immune based assays . 26 3.3.1 Enzyme linked immuno-sorbent assay (ELISA). 26 3.3.2 Proteome Profiler Array . 26 3.3.3 Fluorescence Activated Cell Sorting . 26 3.3.4 Immunocytochemistry . 27 3.4 Transcriptomics and quantitative PCR . 27 3.5 ROC plot analyses . 28 3.6 Gene silencing . 28 3.7 Analysis of hs sulfation patterns . 28 3.8 Functional assays . 28 3.8. 1 Proliferation assay . 28 3.8. 2 Cell cycle distribution . 28 3.8. 3 Apoptosis assay . 29 3.8. 4 Chemtoaxis assay . 29 3.8. 5 Wound healing assay . 29 3.8. 6 Tube formation assay . 29 4 Results and discussion . 30 4.1 Paper I . 30 4.2 Paper II . 31 4.3 Paper III . 32 4.4 Paper IV . 33 5 General conclusion . 36 5.1 Major findings . 36 6 Future perspective . 37 7 Acknowledgements . 38 8 References . 41 LIST OF ABBREVIATIONS CDK Cyclin-dependent kinase