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Fisiopatologia dell’Angiogenesi
oltre il VEGF
Giampaolo Tortora
Cattedra di Oncologia Medica e
U.O.C. di Oncologia Medica
“Policlinico G.B. Rossi“
Azienda Ospedaliera Universitaria Integrata
Verona
Development of Anti-angiogenic drugs in BC
1997
1st Phase 1
study of Bev
initiated
1995
1998
1st Phase 1/2
study of Bev in
MBC initiated;
modest single
agent activity
2003
1st negative
Phase 3 Bev
trial reported in
MBC
2000
2003
Bev ↑ OS in
mCRC leading
to FDA approval
in 2004
2005-2006
TKIs sorafenib &
sunitinib FDAapproved in RCC
2005
2005
Phase 1/2 trials of
sorafenib & sunitinib
reported with modest
single agent activity in
MBC; RCTs with
chemoRx initiated
2005
positive open-label
Phase 3 Bev trial in
MBC reported (E2100)
1st
2010
2008
Bev granted
accelerated FDAapproval with 1st
line paclitaxel in
MBC
The cardiovascular
system
The lymphatic “immuno”
vascular system
The two
vascular systems
L’Angiogenesi è un fenomeno complesso e multifattoriale
I fattori proangiogenici sono molto numerosi :
•
•
•
•
•
•
•
VEGFs
PlGF
bFGFs
HGF
EGFs
IL-8
PDGF
• IGF-I
• TGF-a
• TGF-b
• TNF-a
• GM-CSF
• Angiopoietins
• Angiogenin
Alla neo-angiogenesi partecipano molti tipi cellulari diversi che acquisiscono
la capacità di produrre fattori angiogenici e/o di originare o “trasformarsi” in
cellule endoteliali o simil-endoteliali.
Binding del VEGF ai recettori
VEGFR-1/Flt-1
ANGIOGENESI
VEGFR-2/KDR
VEGFR-3/Flt-4
LINFANGIOGENESI
Binding of different isoforms of VEGF to VEGF-R
Hicklin and Ellis, JCO 2005
VEGFR signalling pathways
PKC, PI3K and Akt are involved in angiogenesis
Angiogenesis
Adapted from Graff J, et al. Cancer Res. 2005;65:7462-7469
Proliferation
VEGF and breast cancer
 VEGF
expression and MVD is increased in breast
cancer
 Increased
VEGF levels in breast cancer correlates with
poor clinical outcome
 Anti-VEGF
treatment inhibits breast cancer growth
Brown, et al. Hum Pathol 1995;
Borgstrom, et al. Anticancer Res 1999;
Linderholm, et al. JCO 2000
HYPOXIA AND ANGIOGENESIS IN BREAST CANCER





Hypoxia related to down regulation of ER
Selection of more aggressive phenotype
Radiation resistance
Independent of all other factors in prognosis
Low oxygen tension is associated with increased
metastasis and decreased survival of patients
45
46
Log Rank
(Mantel-Cox)
Breslow
(Generalized
Wilcoxon)
Chi-Square
df
Sig.
10.9
1
.001
11.2
1
.001
[Affymetrix array-C Sitiriou]
Concentration of VEGF in Conditioned Media of
MCF-7 Neo and MCF-7 HER-2/neu Cells
HER2-overexpressing breast cancer
cells exhibit increased angiogenesis
compared with control cells
3,00E-05
MCF-7 Neo
MCF-7 HER-2/neu
HER2-negative
2,50E-05
1,90E-05
1,86E-05
VEGF (ng/cell)
2,00E-05
1,50E-05
8,78E-06
9,26E-06
1,00E-05
5,96E-06
6,12E-06
6,41E-06
5,30E-06
5,00E-06
0,00E+00
1 nm Control
Slamon D.
1 nm HRG
10 nm Control
10 nm HRG
HER2-positive
VEGF Polymorphism in breast cancer
In E2100 Bev Arm, Improved Survival by Genotype
AA Genotype at VEGF-2578 and -1154 Confers OS Advantage
VEGF-2578
VEGF-2578 AA
N=363
tumor tissue
VEGF-1154 AA
VEGF-1154
E2100, Schneider, B. P. et al. J Clin Oncol; 26:4672-4678; 2008
VEGFs and VEGFRs
Blocking antibodies to VEGFR-3 and TKI as inhibitors
Blocking growth factor binding is inefficient at high ligand
concentrations
Tvorogov et al., Cancer Cell 2010
Helena Schmidt, K. Alitalo
VEGF IS DIFFERENTIALLY EXPRESSED IN THE
VARIOUS GROWTH PHASES
Breast cancer
Colon cancer ?
Modificato sulla base di :
Sund M et al., GASTROENTEROLOGY 2005;129:2076–2091 ; Relf M et al., Cancer Res 1997;57:963–969; Arteaga et al..
Theoretical pathways
by which VEGF-targeted
therapies can increase
tumor aggressiveness
in
Potential
mechanisms
of
resistance
to
VEGF
inhibitors
preclinical models
©2009 by American Association for Cancer Research
Ellis L M, Hicklin D J Clin Cancer Res 2009;15:7471-7478
Angiogenic signaling network: A gene regulatory network constructed from
inversely regulated proangiogenic genes.
Abdollahi A, Transcriptional network governing the angiogenic switch in human pancreatic cancer. PNAS 104: 12890-12895, 2007
Metabolic stress during tumor development
tumors experiencing metabolic stress
Jones R. and Thompson C, Genes & Development 2009
mTOR integra i segnali di nutrienti
e fattori di crescita
Glucosio
 mTOR è un sensore per la disponibilità
di amminoacidi, rifornimenti metabolici
GLUT 1
ed energia
AMPK
PI3K
TSC1
 I depositi di nutrienti e di energia sono
ATP
Segnale di
TSC2
essenziali per la sintesi proteica, la
Akt
Aminoacidi
crescita
crescita cellulare e la sopravvivenza.
LAT
mTOR
 L’ attivazione di mTOR può aumentare
l’espressione dei trasportatori dei
nutrienti,
 Aumentando l’accesso cellulare ai
Sintesi proteica
rifornimenti metabolici, mTOR può
sostenere la crescita cellulare
Bioenergia
Proliferazione cellulare
Glucosio
ATP
Angiogenesi
Interazione vasi- tumore
Folkman J et al., Nature Reviews Cancer, 2007
Viable CEPS in non tumor-bearing
BALBC mice after chemotherapy
 Patients who received chemotherapy exhibited significant
increases in circulating VEGF-A, G-CSF and SDFα levels.
 The SDF-1α plasma levels were significantly induced in
taxane-treated patients.
(Furstenberger, G et al. 2006)
Circulating Bone Marrow–Derived cell populations that
stimulate or amplify tumor angiogenesis.
Kerbel R, New England Journal Medicine,358: 2039-2049, 2008.
potential to differentiate to endothelial
cells and contribute to tumor vasculature
The various hematopoietic (CD45+)
cell types have a perivascular
location with respect to the tumor
neovasculature, whereas
the CD45− endothelial progenitor
cells can become incorporated into
the lumen of a growing blood vessel
and differentiate into mature
endothelial cells.
In recent preclinical studies,
neutrophils have also been shown to
contribute to the induction of tumor
angiogenesis.
53 F4/80, a pan macrophage cell-surface markerM; CXCR4, CXC chemokine receptor 4, RBCCs, recruited bone marrow–derived circulating cells,
VE-cad vascular endothelial-cell cadherin (an adhesion molecule)
Switching in angiogenesis:
the two-faces of microenvironment
Noonan DM, Cancer Metastasis Rev.2007
Macrophages, angiogenesis and cancer
Attract monocyte that
differentiate into TAM
TAM
produce
HIF-1-2
Lee et al. J. Cancer Mol. 2(4): 135-140, 2006
Circulating Bone Marrow–Derived cell populations that
stimulate or amplify tumor angiogenesis.
Kerbel R, New England Journal Medicine,358: 2039-2049, 2008.
potential to differentiate to endothelial
cells and contribute to tumor vasculature
The various hematopoietic (CD45+)
cell types have a perivascular
location with respect to the tumor
neovasculature, whereas
the CD45− endothelial progenitor
cells can become incorporated into
the lumen of a growing blood vessel
and differentiate into mature
endothelial cells.
In recent preclinical studies,
neutrophils have also been shown to
contribute to the induction of tumor
angiogenesis.
53 F4/80, a pan macrophage cell-surface markerM; CXCR4, CXC chemokine receptor 4, RBCCs, recruited bone marrow–derived circulating cells,
VE-cad vascular endothelial-cell cadherin (an adhesion molecule)
BM cells arrive to sites of future
metastasis prior to tumor cells
The experiments of Lyden and the
concept of “premetastatic niche”
They find that cells derived from the bone
marrow (green) precede tumour cells (red) to
the lung, the site of metastasis.
The bone marrow cells create a proposed ‘premetastatic niche’, and the tumour cells join them
to form a metastasis (yellow; in fluorescence
microscopy, green overlaid with red produces a
yellow colour).
Kaplan, R. N. et al. Nature 438, 820–827 (2005).
Steeg P. Nature 438, Editorial 750-751 (2005).
Arrival of VEGFR1+ BM and
VEGFR2+ Endothelial Cells
140
120
cells/100x field
100
80
VEGFR1+
VEGFR2+
60
40
20
0
Day 8
Day 12
Day 14
Day 16 Day 24
Courtesy of David Lyden
Accelerated Metastasis after Short-Term Treatment
with a Potent Inhibitor of Tumor Angiogenesis
No treatment
Rx before implantation
Rx after implantation
Accelerated experimental metastasis and
decreased survival after short-term sunitinib
treatment before and after Intravenous
Tumor Inoculation 231/LM2-4LUC+
Lobos JBL… Kerbel, Cancer Cell 15, 232–239, March 2009
Change in the paradigm of antiangiogenic therapy
• NO to treatments that reduce the number of vessels, cause
hypoxia and consequent angiogenic factors (VEGF)
production, thus favouring tumour regrowth.
• YES to treatments that normalize vessels without affecting
their number and the oxygenation.
Endothelial cells
“sense” O2
and have reduced
oxygen consume.
Jain R, Science 2005; 307: 58
Reduced responsiveness to anti-VEGF-A therapy following co-injection of
CD11b+Gr1+ (but not CD11b-Gr1-) cells from VEGF-independent tumors
CD11b+
Gr1+
100
CD11b+Gr1+
90
* **
* **
+ ++
3500
+ ++
Legend
3000
Cell Line
Mean Tumor Volume (mm3)
Frequency Among GFP+ Cells
60
50
40
30
20
2500
2000
BMMNCs
EL4
CD11b+Gr1+
B16F1
EL4
CD11b-Gr1-
B16F1
LLC
CD11b+Gr1+
B16F1
LLC
CD11b-Gr1-
B16F1
None
B16F1
80
70
Primed Mice
*
*
+
1500
+
*
1000
+
500
10
0
TIB6
B16F1
EL4
LLC
TIB6
B16F1
EL4
LLC
0
1
Anti-VEGF
4
7
10
13
17
21
Control
Refractoriness to anti-VEGF-A is not mediated by the
VEGFR-1 specific ligands VEGF-B or PlGF
Shojaei et al. Nature Biotechnol., 8:911-20, 2007
Anti-Bv8 antibodies suppress tumor angiogenesis and growth
HM7 tumor
Anti-VEGF
Control
Anti-Bv8
Additive effects of anti-VEGF and anti-Bv8 in treating established
tumors
Control
Anti-Bv8
Anti-VEGF
Combination
1600
2500
HM7
Mean Tumour Volume (mm3)
Mean Tumour Volume (mm3)
2000
1200
800
*
*
400
*
*
*
*
2000
A673
Control
Anti-Bv8
Anti-VEGF
Combination
1500
1000
*
500
*
0
*
*
*
*
*
*
*
0
7
11
14
17
21
24
Days
31
34
37
41
12 15 18 21 24 27 31 34 37 41 44 47 51 54
Days
Shojaei et al., Nature 450:825-831, 2007