PhD program

Molecular Signal Tranduction

TUMOR ANGIOGENESIS

Erhard Hofer

Department of Vascular Biology and Thrombosis ResearchCenter for Biomolecular Medicine and PharmacologyMedical University of Vienna

Part I: Overview of vessel formation

1- Angiogenesis and vasculogenesis

2- Important factors and receptors

3- VEGF receptor signaling

4- Tumor angiogenesis

5- Anti-angiogenesis therapies

Literature:

Books:

B. Alberts et al., Molecular Biology of the Cell,5th Edition, Taylor and Francis Inc., 2007Pg. 1279-1283

R.A. Weinberg, The Biology of Cancer, Garland Science, 2007Pg. 556-585

Tumor Angiogenesis - Basic mechanisms and Cancer Therapy, D. Marme, N. Fusenig, ed.Springer Verlag 2008

Angiogenesis - From basic science to clinical applicationN. Ferrara, ed.CRC Press, Taylor&Francis Group, 2007

Nature Insight Angiogenesis G.D. Yancopoulos et al. (2000). Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248.

Angiogenesis Focus, Nature Med 9, June 2003Peter Carmeliet, Angiogenesis in Health and DiseaseNapoleone Ferrara et al., The biology of VEGF and its receptorsRakesh K. Jain, Molecular regulation of vessel maturationShanin Rafii and David Lyden, Therapeutic stem and progenitor celltransplantation for organ vascularization and regenerationChristopher W. Pugh and Peter J. Ratcliffe, Regulation of angiogenesis by hypoxia: role of the HIF system

Angiogenesis, Nature Reviews Cancer 3, June 2003 Gabriele Bergers and Laura E. Benjamin, Tumorigenesis and the angiogenic switch

Literature:

Reviews:

C.J. Schofield and P.J. Ratcliffe. Oxygen sensing by HIF hydroxylases.Nature Rev. Mol. Cell Biol. 5, 343-354 (2004)

Nature Insight Angiogenesis, Vol. 438, pg. 931-974, December 2005Carmeliet, Angiogenesis in life, disease and medicine Coultas, Endothelial cells and VEGF in vascular development Alitalo, Lymphangiogenesis in development and human disease Greenberg, From angiogenesis to neuropathology Gariano, Retinal angiogenesis in development and disease Ferrara, Angiogenesis as a therapeutic target

P. Carmeliet and M. Tessier-Lavigne, Common mechanisms of nerve and blood vessel wiring, Nature 436, 195-200 (2005)

J. Folkman, Angiogenesis: an organizing principle for drug discovery ?Nature Reviews Drug Discovery 6, 273-286 (2007)

Role of notch:Adams and Alitalo, Molecular regulation of angiogenesis and lymphangiogenesis, Nature Rev Mol Cell Biol 8, 464-478 (2007)Germain et al., Hypoxia-driven angiogenesis, Curr Opinion in Hematol 17 (2010)

Guidance cues:Larrivee et al., Guidance of vascular development: Lessons from the nervous system, Circulation Research 104, 428-441 (2009)Gaur et al., Role of class 3 semaphorins and their receptors in tumor growth and angiogenesis, Clin Cancer Res 15, 6763-70 (2009)

Unterlagen:

http://mailbox.univie.ac.at/erhard.hoferStudent point, Vorlesungsunterlagen

erhard.hofer@univie.ac.at

Structure of vessels and capillaries

Monocellular layer of endothelial cellsSmall artery:

Capillary: endothelial cell, basal lamina, pericytes

Angiogenesis:Sprouting of cells from mature endothelial cells of the vessel wall

Mouse cornea:wounding induces angiogenesis,chemotactic response toangiogenic factors

(secretion of proteases, resolution ofBasal lamina, migration towards Chemotactic gradient, proliferation,Tube formation)

VEGF is factor largely specific for endothelial cells,bFGF can also induce, not specific for EC)

Sprouting towards chemotactic gradient: VEGF

Hypoxia - HIF - VEGFevery cell must be within 50 to 100 m of a capillary

HIF: hypoxia inducible factorVEGF: vascular endothelial growth factor

VEGF-gene:Regulated by HIF,HIF is continously produced,ubiquitinylated, degraded in proteasome,therefore low concentration;

Ubiquitinylation dependent onHippel-Lindau tumor suppressor(part of an E3 ubiquitin-ligase complex)

HIF1is modified by a prolyl hydroxylase,then better interaction with vHL protein, high turnover;Hydroxylase is regulated by O2

Von Hippel-Lindau Tumor Suppressor, HIF and VEGF

capillaries sprouting in the retina of an embryonic mouse

capillary lumen opening up

behind the tip cell(red dye injected)

Vasculogenesis

Formation of vessels by differentiation of cells from angioblasts in the yolk sac of the embryo:

Is differentiation and proliferation of endothelial cells in a non-vascularized tissue

Leads to formation of a primitive tubular network

Has to undergo angiogenic remodeling to stable vascular system

Hemangioblast Angioblast EC

Postnatal vasculogenesis

Factors and receptors

Endothelium-specific factors:VEGF family: 5 factorsAngiopoietin family : 4 factorsEphrin family : at least 1 factor

Non EC-specific factors :bFGFPDGFTGF-

VEGF/VEGFR:VEGF-A: initiation of vasculogenesisand sprouting angiogenesis,Immature vessels,Vascular permeability factor,Haploid insufficiency in k.o. mice,

PlGF: remodeling of adult vessels VEGF-B: heart vascularization ?VEGF-C: lymphatic vesselsVEGF-D: lymphatic vessels ?

VEGFR-2: growth and permeabilityVEGFR-1: negative role ?, decoy receptor,

synergism with VEGFR-2 in tumor angiogenesis VEGFR-3: lymphatic vessels

VEGF/VEGFR family

Figure 13.31 The Biology of Cancer (© Garland Science 2007)

Network of lymphatic vessels (red) and capillaries (green):Lymphatic vessels are larger, not supported by underlying mural cells

Differential signaling by tyr kinase receptors

gene regulation

proliferationvasculogenesisangiogenesis

Y799

Y820

Y925

Y936

Y951Y994

Y1006

Y1052Y1057

Y1080Y1104

Y1128Y1134

Y1175Y1212Y1221

Y1303Y1307

Y1317

P38, src (vascular leakage?)

TSAd (migration)

PI-3 kinase (survival)

PLC-

EC “specific” factors/receptors:

VEGFR1 VEGF-A, PLGF

VEGFR2 VEGF-AVEGFR3 VEGF-CTIE1 ?TIE2 ANG1,2

VEGFR2

Sakurai et al.PNAS 2005

82 of the most strongly VEGF-regulated genes (over 5-fold) compared to EGF and IL-1 induction

VEGF + EGF + IL-1 cluster

VEGF + IL-1 cluster

VEGF + EGF cluster

VEGF cluster

Overlapping and specific gene repertoiresof VEGF, EGF and IL-1

About 60 genes reproducibly induced by VEGF over 3-fold

VEGF-induced genes overlap to a large degreewith IL1-induced genes (50-60 %)

20 % of genes are preferentially induced by VEGF

VEGFEGF

IL-160%20%

20%

Signaling by receptors of endothelial cells

PPLC-

1175

P

1175

P

Ca++ PKC

Calcineurin RafMEK/ERK

EGR-1NFAT

P951

PI3K

Akt

TsAd

p38

Actincytoskeleton

Grb2

Ras

RafMEK/ERK

EGR-1

MyD88

IKK/IKK/IKK

IBNFB

NFB

gene regulation survivalmigration

inflammation angiogenesis

permeabilityproliferation

IL-1R VEGFR-2 EGFR

Hofer E., Schweighofer B. Signaling transduction induced in endothelial cells by growth factor receptors involved in angiogenesis. Thrombosis ang haemostasis 2007

IL-1 VEGF-A EGF

Guidance molecules in endothelial tip cell attraction and repulsion

Eichmann A, Curr Opin Neurobiol. 2005

Carmeliet P, Nature. 2005

Angiopoietins und Tie Receptors:

Ang1: remodeling and maturationQuiescence and stabilityResistance to permeability,Supports interaction with other cells and matrix,Vessel size (VEGF number of vessels),Repair of damaged vessels

Ang2: natural antagonist,Overexpression similar Ang-1 k.o. oder Tie-2 k.o.,Destabilization signal for initiation of vascular remodelingEither regression or increased VEGF sensitivityAng2 is induced in tumors

Ang3: ?Ang4: ?

Tie2: binds Ang1-4

Tie1: ?

Ephrins und Eph-Receptors:

Largest family of growth factor receptors,Relevant for vascular system:Ephrin B2/ Eph B4 : remodeling and maturation Different for early arterial (Ephrin B2) and venous vessels (EphB4),Hypothesis: role for fusion of arterial/venous vessels

1-Sprouting

3-incorporation of BM-derived precursors

5-Lymphangiogenesis

Growth of tumor vessels

2-Intussusceptive growth

4-Cooption of existing vessels

Role of VEGF and Ang2 for tumor angiogenesis,VEGF-blockade is promising for anti-ngiogenesis therapy

Concept 2: many tumors “home in” onto vessels, occupate existing vessels,Vessel produces Ang2, first tumor regression, then VEGF production by tumor

Concept 1: non-vascularized Tumor

Figure 13.32a The Biology of Cancer (© Garland Science 2007)

Recruitment of capillaries by an implanted tumor

Figure 13.34a The Biology of Cancer (© Garland Science 2007)

Chaotic organization of tumor-associated vasculature

Structure and function of tumor vessels:

Chaotic architecture and blood flowTherefore hypoxic and acidic regions in tumorPermeability strongly increased

fenestraeenlarged Junctions

No functional lymphatics inside the tumorenlarged in surrounding,increases metastasis

Mosaic vessels

Abnormale endothelium

Figure 13.33 The Biology of Cancer (© Garland Science 2007)

Tumor vessel is only partially overlaid by pericytes and SMC

Figure 13.37 The Biology of Cancer (© Garland Science 2007)

The Rip-Tag model of islet tumor cell progressionTransgene: SV40 large and small T transcription driven by insulin promoter

Transcription in b-cells of islets of Langerhans

Figure 13.38b The Biology of Cancer (© Garland Science 2007)

The angiogenic switch and recruitment of inflammatory cells

Figure 13.49 The Biology of Cancer (© Garland Science 2007)

Heterotypic interactions as targets for therapeutic intervention

Inhibition of tumor angiogenesis

1-Bevacizumab 2-VEGF-trap

3-Pegaptinib

(Combination with 5-fluorouracil forcolorectal cancer)

(Macular degeneration)

4

5- SU11248 Bay43-9006

6- downstream Signals ?

BevacizumabColorectal cancerPhase IIICombination therapy

Hurwitz et al. 2004Mass et al. 2004

IFL: Irinotecan5-fluorouracilLeucovorin

Median survival benefitof two trials (2004):3.7-4.7 months

Gentherapien:

rAdenovirenrRetroviren

Targeting of viruses to tumors, tumor endothelium

Targeting of liposomes to tumors, tumor endothelium

Oncolytic viruses

BM progenitor cells home to tumor vasculature

Next meeting in Zürich, June 15-18, 2011organized by Michael Detmar, ETH

Ralf Adams, Max-Planck-Institute, Münster, Germany

Kari Alitalo, University of Helsinki, Finland

Hirofumi Arakawa, National Cancer Center Research Institute, Tokyo, Japan

Hellmut Augustin, German Cancer Research Center, Heidelberg, Germany

Roy Bicknell, University of Birmingham, UK

Georg Breier, Technical University Dresden, Germany

Peter Carmeliet, Catholic University of Leuven, Belgium

Michael Detmar, Swiss Federal Institute of Technology Zurich, Switzerland

Anna Dimberg, Uppsala University, Sweden

Anne Eichmann, INSERM U833, College de France, Paris, France

Britta Engelhardt, University of Bern, Switzerland

Napoleone Ferrara, Genentech Inc., San Francisco, USA

Holger Gerhardt, London Research Institute, Cancer Research UK

Dontscho Kerjaschki, Medical University of Vienna, Austria

Alexander Koch, Genentech Inc., San Francisco, USA

Donald McDonald, University of California, San Francisco, USA

Gera Neufeld, Israel Institute of Technology, Haifa, Israel

Jaques Pouyssegur, Institute of Developmental Biology and Cancer, Nice,

France

Masabumi Shibuya, University of Tokyo, J apan

Dietmar Vestweber, Max-Planck-Institute, Münster, Germany