PhD program

Molecular Signal Tranduction

TUMOR ANGIOGENESIS
Erhard Hofer
Department of Vascular Biology and Thrombosis Research
Center for Physiology and Pharmacology
Medical 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., 2007
Pg. 1279-1283
R.A. Weinberg, The Biology of Cancer,
Garland Science, 2007
Pg. 556-585
Tumor Angiogenesis - Basic mechanisms and Cancer Therapy,
D. Marme, N. Fusenig, ed.
Springer Verlag 2008
Angiogenesis - From basic science to clinical application
N. Ferrara, ed.
CRC Press, Taylor&Francis Group, 2007

Literature:
Reviews:
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 2003

Peter Carmeliet, Angiogenesis in Health and Disease
Napoleone Ferrara et al., The biology of VEGF and its receptors
Rakesh K. Jain, Molecular regulation of vessel maturation
Shanin Rafii and David Lyden, Therapeutic stem and progenitor cell
transplantation for organ vascularization and regeneration
Christopher 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

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 2005

Carmeliet, 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.hofer
Student point, Vorlesungsunterlagen
erhard.hofer@univie.ac.at

Structure of vessels and capillaries

Small artery: Monocellular layer of endothelial cells

Capillary: endothelial cell,

basal lamina, pericytes

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

(secretion of proteases, resolution of

Basal lamina, migration towards
Chemotactic gradient, proliferation,
Tube formation)
VEGF is factor largely specific for
endothelial cells,
bFGF can also induce,
not specific for EC)

Mouse cornea:
wounding induces
angiogenesis,
chemotactic
response to
angiogenic factors

Sprouting towards chemotactic gradient: VEGF

Hypoxia - HIF - VEGF

every cell must be within 50 to 100 m of a capillary

HIF: hypoxia inducible factor

VEGF: vascular endothelial growth factor

Von Hippel-Lindau Tumor Suppressor, HIF and VEGF

VEGF-gene:
Regulated by HIF,
HIF is continously produced,
ubiquitinylated,
degraded in proteasome,
therefore low concentration;
Ubiquitinylation dependent on
Hippel-Lindau tumor
suppressor
(part of an E3 ubiquitin-ligase
complex)
HIF1 is modified by a
prolyl hydroxylase,
then better interaction with
vHL protein, high turnover;
Hydroxylase is regulated by O2

capillaries sprouting in
the retina of an embryonic mouse

capillary lumen opening up

behind the tip cell
(red dye injected)

Network of lymphatic vessels (red) and capillaries (green):

Lymphatic vessels are larger, not supported by underlying mural cells

Figure 13.31 The Biology of Cancer ( Garland Science 2007)

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

Angiogenesis - Vasculogenesis

Adams, R. and Alitalo, K. 2007, Nat Rev Mol Cell Biol.

Postnatal vasculogenesis
Hemangioblast

Angioblast

EC

Generation of Circulating endothelial progenitor

cells (CEPs) derived from cord blood

A.Total CB-MNC
B. CD133 separation

CD133

Factors and receptors

Endothelium-specific factors:
VEGF family: 5 factors
Angiopoietin family : 4 factors
Ephrin family : at least 1 factor
Non EC-specific factors :
bFGF
PDGF
TGF-

VEGF/VEGFR family

VEGF/VEGFR:
VEGF-A: initiation of vasculogenesis
and 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 vessels
VEGF-D: lymphatic vessels ?
VEGFR-2: growth and permeability
VEGFR-1: negative role ?, decoy receptor,
synergism with VEGFR-2 in
tumor angiogenesis
VEGFR-3: lymphatic vessels

Angiopoietins und Tie Receptors:

Ang1: remodeling and maturation

Quiescence and stability
Resistance 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 remodeling
Either regression or increased VEGF sensitivity
Ang2 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

Differential signaling by tyr kinase receptors

EC specific factors/receptors:

VEGFR2

VEGFR1

VEGF-A, PLGF

VEGFR2

VEGF-A

VEGFR3
TIE1
TIE2

VEGF-C
?
ANG1,2

Y799
Y820

P38, src (vascular leakage?)

Y925
Y936

Y951

TSAd (migration)

Y994
Y1006
Y1052
Y1057

PI-3 kinase (survival)

Y1080
Y1104

gene regulation

Y1128
Y1134

Y1175
Y1212
Y1221
Y1303
Y1307
Y1317

PLC-

proliferation
vasculogenesis
angiogenesis

Sakurai et al.
PNAS 2005

82 of the most strongly VEGFregulated genes (over 5-fold) compared

to EGF and IL-1 induction
VEGF + IL-1 cluster

VEGF + EGF + IL-1 cluster

VEGF + EGF cluster

VEGF cluster

Overlapping and specific gene repertoires

of VEGF, EGF and IL-1

VEGF
20%

EGF
20%
60%

IL-1

About 60 genes reproducibly induced by VEGF over 3-fold

VEGF-induced genes overlap to a large degree
with IL1-induced genes (50-60 %)
20 % of genes are preferentially induced by VEGF

Signaling by receptors of endothelial cells

IL-1

VEGF-A
IL-1R

EGF

VEGFR-2

EGFR

p38
P
PLC-

MyD88

951

TsAd

1175

1175

PI3K
IKK/IKK/IKK

Ca++

P
Grb2
Actin
cytoskeleton

PKC

Ras
Akt

IB
NFB
NFB

Calcineurin

NFAT

EGR-1

EGR-1

gene regulation
proliferation
inflammation

Raf
MEK/ERK

Raf
MEK/ERK

survival
migration
permeability
angiogenesis

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

The VEGF cluster:

Part of the VEGF cluster genes is upregulated also by bFGF
EGR3

1600

HLX1

120

10
8

1200

80

VEGF
IL1
bFGF
EGF

800

40

400

0
0

100

200

300

400

0
0

100

200

300

400

100

t (min)

t (min)

200

300

400

t (min)

MEF2C

IGFBP3

mRNA fold induction

mRNA fold induction

NR4A2

VEGF
IL1
bFGF
EGF

0
0

100

200

t (min)

300

400

100

200

300

400

t (min)

Real time RT-PCR

HLX induced genes (microarray)

Guidance molecules in endothelial tip cell

attraction and repulsion

Carmeliet P, Nature. 2005

Eichmann A, Curr Opin

Neurobiol. 2005

Guidance cues
regulating angiogenesis at capillary tip

Larrive, B. et al, Circ Res

2009

VEGF induces HLX and UNC5B

Western blot

RT-PCR

VEGF induces HLX and UNC5B

HLX strongly inhibits angiogenic sprouting

Spheroid Assay

Endothelial spheroid xenografting assay

Three-dimensional structure of the human

neovasculature as assessed by confocal
microscopy using immunofluorescence wholemount staining for hCD31 of a plug implanted
for 20 d (slice 0.78m,zaxis 36.5m).

Overexpression of HLX inhibits vessels

formation in vivo

Endothelial spheroid
xenografting assay

Hypoxic gradient- regulates vessel guidance

Alberts, B. et al, Molecular Biology

of the Cell, 2008

Carmeliet, P. and Jain, R. Nature

2000

Control of angiogenic sprouting

Adams, R. and Alitalo, K. 2007, Nat Rev Mol Cell Biol.

Four families of guidance cues and receptors

Adams , R. and Eichmann, A.

Cold Spring Harb Perspect Biol
2010

Growth of tumor vessels

3-incorporation of
BM-derived precursors
2-Intussusceptive
growth

1-Sprouting

4-Cooption of existing vessels

5-Lymphangiogenesis

Role of VEGF and Ang2 for tumor angiogenesis,

VEGF-blockade is promising for anti-ngiogenesis therapy
Concept 1: non-vascularized Tumor

Concept 2: many tumors home in onto vessels, occupate existing vessels,

Vessel produces Ang2, first tumor regression, then VEGF production by tumor

Recruitment of capillaries by an implanted tumor

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

Chaotic organization of tumor-associated vasculature

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

Structure and function of tumor vessels:

Chaotic architecture and blood flow
Therefore hypoxic and acidic regions in tumor
Permeability strongly increased
fenestrae
enlarged Junctions
No functional lymphatics inside the tumor
enlarged in surrounding,
increases metastasis

Mosaic vessels

Abnormale endothelium

Tumor vessel is only partially overlaid by pericytes and SMC

Figure 13.33 The Biology of Cancer ( Garland Science 2007)

The Rip-Tag model of islet tumor cell progression

Transgene: SV40 large and small T transcription driven by insulin promoter
Transcription in b-cells of islets of Langerhans

Figure 13.37 The Biology of Cancer ( Garland Science 2007)

The angiogenic switch and recruitment of inflammatory cells

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

Heterotypic interactions as targets for therapeutic intervention

Figure 13.49 The Biology of Cancer ( Garland Science 2007)

Inhibition of tumor angiogenesis

(Combination with
5-fluorouracil for
colorectal cancer)

1-Bevacizumab

2-VEGF-trap

3-Pegaptinib
(Macular degeneration)

6- downstream
Signals ?
5- SU11248
Bay43-9006

Bevacizumab
Colorectal cancer
Phase III
Combination therapy
Hurwitz et al. 2004
Mass et al. 2004
IFL:
Irinotecan
5-fluorouracil
Leucovorin
Median survival benefit
of two trials (2004):
3.7-4.7 months

Gentherapien:
rAdenoviren
rRetroviren
Targeting of viruses to tumors, tumor endothelium
Targeting of liposomes to tumors, tumor endothelium
Oncolytic viruses
BM progenitor cells home to tumor vasculature

http://www.pharma.ethz.ch/ec8zurich2011