Signalling and expression of the Ang-Tie pathway in tumor vasculature Anita Lampinen Research Programs Unit Translational Cancer Biology Program & Doctoral Programme in Biomedicine Faculty of Medicine University of Helsinki Finland Academic dissertation To be presented for public examination with the permission of the Faculty of Medicine, University of Helsinki, in Lecture Hall 3, Biomedicum Helsinki 1 (Haartmaninkatu 8, Helsinki), on December 15th 2016, at 12 noon. Thesis supervisor Docent, Ph.D. Pipsa Saharinen Wihuri Research Institute Translational Cancer Biology Program Research Programs Unit Faculty of Medicine University of Helsinki Finland Reviewers appointed by the Faculty Prof. M.D., Ph.D. Peppi Karppinen Faculty of Biochemistry and Molecular Medicine University of Oulu Finland and Prof. M.D., Ph.D. Tom Böhling Medicum Haartman Institute Department of Pathology University of Helsinki Finland Opponent appointed by the Faculty Associate Prof., Docent, Ph.D. Anna Dimberg Department of Immunology, Genetics and Pathology Uppsala University Sweden ISBN 978-951-51-2758-7 (paperback) ISBN 978-951-51-2759-4 (PDF) ISSN 2342-3161 http://ethesis.helsinki.fi Unigrafia Oy Helsinki 2016 Sisu alkaa siellä, missä sinnikkyys ja periksiantamattomuus loppuvat. Sisu on henkisen kestävyyden ja rikkoutumattomuuden toinen aalto, sekä toiminnan ääritilanteessa mahdollistava psyykkinen voimavara. -Emilia Lahti CONTENTS LIST OF PUBLICATIONS ......................................................................................... 8 ABBREVIATIONS ...................................................................................................... 9 TIIVISTELMÄ .......................................................................................................... 11 ABSTRACT ............................................................................................................... 13 INTRODUCTION ...................................................................................................... 15 LITERATURE REVIEW ........................................................................................... 17 1. Blood vasculature ............................................................................................ 17 1.1. Structure and functions of the blood vascular system ............................. 17 1.2. Growth factor regulation of vascular endothelial cells ............................ 17 1.2.1. VEGFs and VEGFRs ........................................................................ 17 1.2.2. Angiopoietin-Tie system .................................................................. 20 1.2.3. Regulation of Ang-Tie signalling by Tie1 and integrins .................. 23 1.3. Formation of the blood vascular system .................................................. 24 1.3.1. Molecular regulation of angiogenesis .............................................. 24 1.3.2. Tip and stalk cells ............................................................................. 25 1.4. Tumor angiogenesis and features of the tumor vasculature..................... 26 1.4.1. VEGFs and Ang2 in tumor angiogenesis ......................................... 27 1.5. Regulation of vascular permeability ........................................................ 29 1.5.1. Endothelial cell-cell junctions .......................................................... 29 1.5.2. Inflammation .................................................................................... 32 2. Renal cell carcinoma (RCC) ........................................................................... 33 2.1. Epidemiology and pathology ................................................................... 33 2.2. von Hippel-Lindau (VHL) and hypoxia .................................................. 34 2.3. Metastatic renal cell carcinoma (mRCC) ................................................. 34 2.4. Microvessel density ................................................................................. 35 2.5. Therapeutic options .................................................................................. 35 2.5.1. Immunotherapy ................................................................................. 35 2.5.2. Tyrosine kinase inhibitors (TKIs) .................................................... 36 2.5.3. Mammalian target of rapamycin (mTOR) inhibitors ....................... 38 2.6. Prognostic and predictive biomarkers of renal cell carcinoma ................ 40 AIMS OF THE STUDY ............................................................................................. 42 PATIENTS, MATERIALS AND METHODS .......................................................... 43 5 1. Patients and treatments ................................................................................... 43 1.1. Patients in publication I ........................................................................... 43 1.2. Patients in publication II .......................................................................... 43 2. Materials ......................................................................................................... 43 2.1. Cell lines .................................................................................................. 43 2.2. Primary antibodies ................................................................................... 44 2.3. Lentiviruses ............................................................................................. 45 2.4. Retroviruses ............................................................................................. 45 2.5. Recombinant proteins .............................................................................. 45 2.6. Mouse lines and viruses .......................................................................... 45 2.7. Cell culture (III, IV) ................................................................................ 46 3. Methods .......................................................................................................... 46 3.1. Cell stimulations (III, IV) ........................................................................ 46 3.2. Viral vector delivery (III, IV) .................................................................. 46 3.3. In vivo treatments (III, IV) ...................................................................... 47 3.4. Cell sorting and flow cytometry (III) ...................................................... 47 3.5. Immunofluorescence staining (III, IV) .................................................... 48 3.6. Immunoprecipitation and Western blot (III) ........................................... 48 3.7. Microscopy, confocal microscopy, live cell imaging and image analysis (I-IV) 48 3.8. FLIM and FRET (III) .............................................................................. 49 3.9. Time-Correlated Single Photon Counting (TCSPC) (III) ....................... 49 3.10. Immunohistochemistry (I, II) ............................................................... 50 3.11. Statistical analyses ............................................................................... 50 RESULTS AND DISCUSSION ................................................................................ 52 1. Tie1 participates in angiopoietin signalling in vivo and in vitro (III) ............. 52 2. Tie1 is required for Angiopoietin-mediated vascular remodelling (III) ......... 52 3. Angiopoietin induced Tie2 phosphorylation and Foxo1 inactivation is impaired in Tie1 deficiency (III) ............................................................................ 53 4. Ang2 acts as a Tie2 antagonist in inflammatory conditions (III) ................... 53 5. Tie1 ectodomain is cleaved in inflammation leading to a loss of agonistic effects of Angiopoietins (III) ................................................................................. 54 6. Direct interactions of Tie1 and Tie2 are induced by angiopoietins (III) ........ 54 6.1. FRET based on acceptor photobleaching ................................................ 54 6.2. Frequence domain FLIM ......................................................................... 55 6 6.3. Summary of FRET and FLIM results ...................................................... 55 7. Ang-Tie signalling is dependent on α β -integrin (III) ................................... 56 5 1 8. Tie1 is required for Ang2 agonist function (III) ............................................. 57 9. Tie2 trafficking is altered in the absence of Tie1 (III) .................................... 57 10. Ang2 modulates endothelial cell-cell junctions in blood vasculature enhancing tumor cell extravasation and metastasis (IV) ........................................ 58 11. Ang2 in renal cell carcinoma (I-II) .............................................................. 59 11.1. Ang2 is expressed in mRCC tumor endothelium and correlates with vascular density (I) .............................................................................................. 59 11.2. High Ang2 expression was correlated with better response to sunitinib treatment (I) ........................................................................................................ 60 11.3. High Ki-67 expression in tumor cells predicted poor prognosis (I) ..... 62 11.4. Angiogenesis and proliferation markers Ki-67 and BCL-2 as long-term prognostic factors in RCC patients (II) ............................................................... 62 11.5. High Ang2 expression correlated with tumor grade, longer survival and low tumor cell proliferation (II) .......................................................................... 62 CONCLUSIONS ........................................................................................................ 64 AKNOWLEDGEMENTS .......................................................................................... 66 REFERENCES ........................................................................................................... 68 7 LIST OF PUBLICATIONS This thesis is based on the following publications and will be referred in the text by their Roman numerals (I-IV). The original publications have been reprinted with the permission of the publisher. I. Rautiola J*, Lampinen A*, Mirtti T, Ristimäki A, Joensuu H, Bono P and Saharinen P. Association of Angiopoietin-2 and Ki-67 Expression with Vascular Density and Sunitinib Response in Metastatic Renal Cell Carcinoma. Plos One 11(4):e0153745, 2016. *Equal contribution. II. Lampinen A, Virman J, Bono P, Luukkaala T, Sunela K, Kujala P, Saharinen P and Kellokumpu-Lehtinen P-L. Novel angiogenesis markers as long-term prognostic factors in renal cell cancer patients. Clinical Genitourinary Cancer 2016 Jul [Epub ahead of print]. III. Emilia A. Korhonen*, Anita Lampinen*, Hemant Giri, Andrey Anisimov, Minah Kim, Breanna Allen, Shentong Fang, Gabriela D’Amico, Tuomas Sipilä, Marja Lohela, Tomas Strandin, Antti Vaheri, Seppo Ylä-Herttuala, Gou Young Koh, Donald M. McDonald, Kari Alitalo# and Pipsa Saharinen#. Tie1 controls angiopoietin function in vascular remodeling and inflammation. Journal of Clinical Investigation. 126(9):3495-510, 2016. * # Equal contribution. IV. Holopainen T, Saharinen P, D’Amico G, Lampinen A, Eklund L, Sormunen R, Anisimov, A, Zarkada G, Lohela M, Heloterä H, Tammela T, Benjamin LE, Ylä-Herttuala S, Leow CC, Koh GY and Alitalo K. Effects of angiopoietin-2-blocking antibody on endothelial cell-cell junctions and lung metastasis. Journal of the National Cancer Institute. 104(6):461-475, 2012. Publication I appears in the doctoral thesis of Dr. Juhana Rautiola (2015, University of Helsinki). Publication II appears in the doctoral thesis of Dr. Juha Virman (2016, University of Tampere). Publication IV appears in the doctoral thesis of Dr. Tanja Holopainen (2016, University of Helsinki). 8 ABBREVIATIONS Ab antibody Akt a serine/threonine kinase Ang1 angiopoietin-1, also termed Angpt1 Ang2 angiopoietin-2, also termed Angpt2 BEC blood vascular endothelial cell BM basement membrane CAIX carbonic anhydrase-9 CBR clinical benefit rate CCD coiled-coil domain ccRCC clear cell renal cell carcinoma Comp cartilage oligomeric matrix protein EC endothelial cell ECM extracellular matrix EGF epidermal growth factor ESAM endothelial cell-selective adhesion molecule ESM-1 endothelial cell specific molecule 1 FAK focal adhesion kinase FLIM fluorescence lifetime imaging microscopy Flk-1 Fetal liver kinase 1 FN III fibronectin type III repeats Foxo1 the Forkhead box protein O1 FRET fluorescence resonance energy transfer HGF hepatocyte growth factor HIF hypoxia-inducible factor ICAM intercellular adhesion protein IF immunofluorescence Ig immunoglobulin IHC immunohistochemistry IL interleukin IP immunoprecipitation JAM junction adhesion molecule KDR kinase insert domain-containing receptor LPS lipopolysaccharide miRNA micro RNA MAPK mitogen activated protein kinase MMP matrix metalloproteinase mRCC metastatic renal cell carcinoma mRNA messenger ribonucleic acid mTOR mammalian target of rapamycin MVD microvessel density NRP-1 neuropilin-1 OS overall survival ORR objective response rate PECAM-1 the platelet endothelial cell adhesion molecule-1 PKC protein kinase C PD progressive disease 9 PDGF plateled-derived growth factor PFS progression-free survival PHD prolyl hydroxylase PlGF placental growth factor PMA phorbol myristate acetate PR partial response pRCC papillary renal cell carcinoma Rap1 Ras-proximate-1 or Ras-related-protein 1 RCC renal cell carcinoma ROCK Rho-associated protein kinase ROI region of interest RTK receptor tyrosine kinase SCD super-clustering domain SD stable disease SGK1 serine/threonine-protein kinase shRNA short hairpin ribonucleic acid SMC smooth muscle cell SNP single nucleotide polymorphism sTie soluble Tie TEM Tie2 expressing monocyte/macrophage TIMP tissue inhibitor of metalloproteinase or TIMP metallopeptidase inhibitor TKI tyrosine kinase inhibitor TNF-α tumor necrosis factor-α TMA tissue microarray VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor VE-PTP Receptor-type tyrosine-protein phosphatase beta VHL von Hippel-Lindau wAMD wet age-related macular degeneration WB Western blot WT wild type ZO-1 zonula occludens-1 10