Signalling and expression of the Ang-Tie pathway in tumor vasculature

96 Pages·2016·1.53 MB·English

by  Anita Lampinen

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
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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

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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).
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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

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