1. 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

2. Development of Anti-angiogenic drugs in BC
2003
1st negative Phase 3 Bev trial reported in MBC
TKIs sorafenib & sunitinib FDA-approved in RCC
2005
1st positive open-label Phase 3 Bev trial in MBC reported (E2100)
1997
1st Phase 1 study of Bev initiated
1995
2000
2005
2010
2003
Bev ↑ OS in mCRC leading to FDA approval in 2004
2005
Phase 1/2 trials of sorafenib & sunitinib reported with modest single agent activity in MBC; RCTs with chemoRx initiated
1998
1st Phase 1/2 study of Bev in MBC initiated; modest single agent activity
2008
Bev granted accelerated FDA-approval with 1st line paclitaxel in MBC

3. The lymphatic “immuno” vascular system
The cardiovascular
system
The lymphatic “immuno” vascular system
The two
vascular systems

4. 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. 4

5. Binding del VEGF ai recettori
VEGFR-3/Flt-4
VEGFR-1/Flt-1
VEGFR-2/KDR
ANGIOGENESI
LINFANGIOGENESI

6. Binding of different isoforms of VEGF to VEGF-R
Hicklin and Ellis, JCO 2005

7. VEGFR signalling pathways

8. PKC, PI3K and Akt are involved in angiogenesis
Proliferation
Adapted from Graff J, et al. Cancer Res. 2005;65: 8

9. VEGF expression and MVD is increased in breast cancer
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 9

10. 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
[Affymetrix array-C Sitiriou]

11. 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
MCF-7 Neo
MCF-7 HER-2/neu
HER2-negative
VEGF (ng/cell)
HER2-positive
Slamon D.

12. VEGF Polymorphism in breast cancer

13. AA Genotype at VEGF-2578 and -1154 Confers OS Advantage
In E2100 Bev Arm, Improved Survival by Genotype
AA Genotype at VEGF-2578 and 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: ; 2008

14. VEGFs and VEGFRs

15. Blocking antibodies to VEGFR-3 and TKI as inhibitors

16. Blocking growth factor binding is inefficient at high ligand concentrations
Tvorogov et al., Cancer Cell 2010

17. Helena Schmidt, K. Alitalo

18. 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..

19. Potential mechanisms of resistance to VEGF inhibitors
Theoretical pathways by which VEGF-targeted therapies can increase tumor aggressiveness in preclinical models
Theoretical pathways by which VEGF-targeted therapies can increase tumor aggressiveness in preclinical models. Preclinical studies have identified four potential mechanisms of resistance to VEGF inhibitors: (1) activation and/or up-regulation of alternative angiogenic signaling pathways by tumor cells; (2) recruitment of bone marrow-derived cells that up-regulate angiogenic pathways; (3) increased pericyte coverage leading to VEGF-independent tumor vasculature; and (4) activation of invasion and/or metastasis mechanisms to supply tumor cells via established vasculature rather than stimulation of new blood vessels. During progression of malignant disease, these mechanisms may play a role in acquired (1) or intrinsic (2) resistance to VEGF- targeted therapy. In situations in which patients initially respond to VEGF-targeted agents, one or more of these resistance mechanisms are up-regulated leading to the eventual failure of therapy. Intrinsic resistance occurs when one or more resistance mechanisms are up- regulated by the natural course of the disease leading to initial failure of VEGF-targeted therapy. Recent studies propose that under some circumstances, VEGF inhibition can lead to an increase in levels of circulating proangiogenic and pro-invasive factors that could potentially lead to an increase in tumor aggressiveness after an initial response to therapy.
©2009 by American Association for Cancer Research
Ellis L M, Hicklin D J Clin Cancer Res 2009;15:

20. 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: , 2007

21. Metabolic stress during tumor development
tumors experiencing metabolic stress
Jones R. and Thompson C, Genes & Development 2009

22. mTOR integra i segnali di nutrienti e fattori di crescita
Glucosio
Glucosio
mTOR è un sensore per la disponibilità di amminoacidi, rifornimenti metabolici ed energia
I depositi di nutrienti e di energia sono essenziali per la sintesi proteica, la crescita cellulare e la sopravvivenza.
L’ attivazione di mTOR può aumentare l’espressione dei trasportatori dei nutrienti,
Aumentando l’accesso cellulare ai rifornimenti metabolici, mTOR può sostenere la crescita cellulare
ATP
GLUT 1
AMPK
PI3K
TSC1
ATP
Segnale di crescita
TSC2
Akt
Aminoacidi
LAT
mTOR
mTOR is a sensor that acts as a biochemical switch, ensuring that adequate supplies of energy and nutrients are available to support cell growth, cell metabolism, and angiogenesis
Nutrients (amino acids, glucose, cholesterol, iron, zinc): mTOR is a sensor of nutrient availability
Amino acids are taken into the cell via transporters located at the surface of the cell. When cell signaling and/or metabolic activity in the cell are increased, there is an increased need for essential amino acids. Cells meet this need by synthesizing more transporters thus allowing uptake of more nutrients
Depending on the tissue type, glucose can be used by the cell as a carbon source to build fatty acids, amino acids, or other macromolecules. Additionally, glucose can be used as a metabolic fuel to obtain the energy required for cell survival
Energy for cell processes is provided by ATP, an energy-storing molecule found in cells. mTOR senses ATP availability in the cell through its regulation by AMPK.
When resources are low (low ATP, low oxygen, low amino acids, and/or low glucose), cells experience nutrient deprivation and a slowed metabolism. In nutrient- and energy-poor environments, there is a relative increase in the activity of AMP kinase (AMPK). The increased activity of AMPK inhibits the mTOR pathway by phosphorylating and activating TSC2, thus ensuring biological processes do not occur in the absence of adequate nutrient and energy resources.
References
Marshall S. Sci STKE. 2006;(346):re7.
Herman and Kahn. J Clin Invest. 2006;116:
Motoshima et al. J Physiol. 2006;574:63-71.
Edinger and Thompson. Mol Biol Cell. 2002;13:
Sintesi proteica
Proliferazione cellulare
Bioenergia
Angiogenesi 22 22

23. Interazione vasi- tumore
Folkman J et al., Nature Reviews Cancer, 2007

24. 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)

25. Circulating Bone Marrow–Derived cell populations that
stimulate or amplify tumor angiogenesis.
Kerbel R, New England Journal Medicine,358: , 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)

26. Switching in angiogenesis: the two-faces of microenvironment
Noonan DM, Cancer Metastasis Rev.2007

27. Macrophages, angiogenesis and cancer
TAM produce
HIF-1-2
Attract monocyte that differentiate into TAM
Lee et al. J. Cancer Mol. 2(4): , 2006

28. Circulating Bone Marrow–Derived cell populations that
stimulate or amplify tumor angiogenesis.
Kerbel R, New England Journal Medicine,358: , 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)

29. 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 ‘pre-metastatic niche’, and the tumour cells join them to form a metastasis (yellow; in fluorescence microscopy, green overlaid with red produces a yellow colour).
Bevacizumab targets VEGF, which, since it was characterised in the 1980s, has been extensively studied, in animal and human models. Its mechanism of action and interaction with receptors is well understood.1–4
VEGF is likely to be the most important promoter of angiogenesis: it directly stimulates angiogenesis by increasing vascular permeability, and mediating endothelial cell migration, proliferation and survival. VEGF is the one growth factor that most consistently found in many conditions associated with angiogenesis.5
Other anti-angiogenic agents are specific only to molecules such as matrix metalloproteinases.6 VEGF actually regulates these secondary molecules,2 so targeting VEGF will include inhibition of these molecules as well.
Many agents (e.g. HER1/EGFR-targeting agents) may indirectly inhibit angiogenesis by regulating VEGF expression. For instance Iressa (gefitinib) suppresses angiogenesis by inhibiting VEGF production through inhibition of EGFR signalling.7
Bevacizumab inhibits all functions of the VEGF ligand. In in-vitro, animal and human studies, treatment with bevacizumab reduces vascular permeability, reduces microvascular density and normalises vasculature, and improves immune response to tumours in mice when given alongside immunotherapy.
Many studies have shown bevacizumab’s activity on vascular endothelial cells, but it also inhibits VEGF activity on non-endothelial cells (e.g. dendritic cells, monocytes).
Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–76.
Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25.
Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653–60.
Gerber H-P, Ferrara N. The role of VEGF in normal and neoplastic hematopoiesis. J Mol Med 2003;81:20–31.
Zhu Z, Bohlen P, Witte L. Clinical development of angiogenesis inhibitors to vascular endothelial growth factor and its receptors as cancer therapeutics. Curr Cancer Drug Targets 2002;2:135–56.
Griffioen AW, Molema G. Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 2000;52:237–68.
Yano S, Yamaguchi M, Dong RP. EGFR tyrosine kinase inhibitor 'gefitinib (Iressa)' for cancer therapy. Nippon Yakurigaku Zasshi 2003;122:491–7.
Kaplan, R. N. et al. Nature 438, 820–827 (2005).
Steeg P. Nature 438, Editorial (2005).

30. Arrival of VEGFR1+ BM and VEGFR2+ Endothelial Cells
Courtesy of David Lyden

31. 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

32. Endothelial cells “sense” O2 and have reduced oxygen consume.
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

33. Reduced responsiveness to anti-VEGF-A therapy following co-injection of CD11b+Gr1+ (but not CD11b-Gr1-) cells from VEGF-independent tumors
Frequency Among GFP+ Cells
CD11b+
Gr1+
CD11b+Gr1+ 10 20 30 40 50 60 70 80 90
100
Anti-VEGF
Control
TIB6
B16F1
EL4
LLC * +
500
1500
2500 1 4 7 10 13 17 21
Legend
Primed Mice
B16F1
Cell Line
EL4
LLC
None
BMMNCs
CD11b+Gr1+
CD11b-Gr1- + *
1000
2000
3000
Mean Tumor Volume (mm3)
3500
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

34. Anti-Bv8 antibodies suppress tumor angiogenesis and growth
HM7 tumor
Anti-VEGF
Anti-Bv8
Control
Additive effects of anti-VEGF and anti-Bv8 in treating established tumors
500
1000
1500
2000
2500 12 15 18 21 24 27 31 34 37 41 44 47 51 54
Days
Control
Anti-Bv8
Anti-VEGF
Combination *
Mean Tumour Volume (mm3)
400
800
1200
1600 7 11 14 17
HM7
A673
Shojaei et al., Nature 450: , 2007