Giampaolo Tortora

Fisiopatologia dell’Angiogenesi oltre il VEGF

Cattedra di Oncologia Medica eU.O.C. di Oncologia Medica

“Policlinico G.B. Rossi“Azienda Ospedaliera Universitaria Integrata

Verona

Development of Anti-angiogenic drugs in BCDevelopment of Anti-angiogenic drugs in BC

1998 1st Phase 1/2

study of Bev in MBC initiated; modest single agent activity

1995

1997 1st Phase 1 study of Bev

initiated

20031st negative

Phase 3 Bev trial reported in

MBC

20051st positive open-label Phase 3 Bev trial in

MBC reported (E2100)

2003Bev ↑ OS in

mCRC leading to FDA approval

in 2004

2005Phase 1/2 trials of

sorafenib & sunitinib reported with modest single agent activity in

MBC; RCTs with chemoRx initiated

2005-2006TKIs sorafenib &

sunitinib FDA-approved in RCC

2000 2005 2010

2008Bev granted

accelerated FDA-approval with 1st line paclitaxel in

MBC

The cardiovascularsystem

The lymphatic “immuno” vascular system

The two vascular systems

L’Angiogenesi è un fenomeno complesso e multifattorialeL’Angiogenesi è un fenomeno complesso e multifattoriale

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.

I fattori proangiogenici sono molto numerosi :

• VEGFsVEGFs• PlGFPlGF• bFGFsbFGFs• HGFHGF• EGFsEGFs• IL-8IL-8• PDGFPDGF

• IGF-I • TGF-• TGF-• TNF-• GM-CSF• Angiopoietins• Angiogenin

Binding del VEGF ai recettoriBinding del VEGF ai recettori

VEGFR-1/Flt-1 VEGFR-2/KDRVEGFR-3/Flt-4VEGFR-3/Flt-4

LINFANGIOGENESIANGIOGENESI

Binding of different isoforms of VEGF to VEGF-RBinding of different isoforms of VEGF to VEGF-R

Hicklin and Ellis, JCO 2005

VEGFR signalling pathwaysVEGFR signalling pathways

PKC, PI3K and Akt are involved in angiogenesisPKC, PI3K and Akt are involved in angiogenesis

Adapted from Graff J, et al. Cancer Res. 2005;65:7462-7469

Proliferation Angiogenesis

VEGF and breast cancerVEGF 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 CANCERHYPOXIA AND ANGIOGENESIS IN BREAST CANCER

45

46

[Affymetrix array-C Sitiriou]

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

6,12E-06

9,26E-068,78E-06

6,41E-06

1,86E-05

1,90E-05

5,30E-06

5,96E-06

0,00E+00

5,00E-06

1,00E-05

1,50E-05

2,00E-05

2,50E-05

3,00E-05

1 nm Control 1 nm HRG 10 nm Control 10 nm HRG

VE

GF

(n

g/c

ell)

MCF-7 NeoMCF-7 HER-2/neu

Concentration of VEGF in Conditioned Media of MCF-7 Neo and MCF-7 HER-2/neu Cells

HER2-negative

HER2-positive

HER2-overexpressing breast cancer cells exhibit increased angiogenesis

compared with control cells

Slamon D.

VEGF Polymorphism in breast cancerVEGF Polymorphism in breast cancer

E2100, Schneider, B. P. et al. J Clin Oncol; 26:4672-4678; 2008

In E2100 Bev Arm, Improved Survival by Genotype

VEGF-2578

VEGF-1154

VEGF-2578 AA

VEGF-1154 AA

N=363 tumor tissue

AA Genotype at VEGF-2578 and -1154 Confers OS Advantage

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 VEGF IS DIFFERENTIALLY EXPRESSED IN THE VARIOUS GROWTH PHASES VARIOUS GROWTH PHASES

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..

Breast cancerBreast cancerColon cancer ?Colon cancer ?

Theoretical pathways by which VEGF-targeted therapies can increase tumor aggressiveness in preclinical models

©2009 by American Association for Cancer Research Ellis L M, Hicklin D J Clin Cancer Res 2009;15:7471-7478

Potential mechanisms of resistance to VEGF inhibitorsPotential mechanisms of resistance to VEGF inhibitors

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 developmentMetabolic stress during tumor development

Jones R. and Thompson C, Genes & Development 2009

tumors experiencing metabolic stress

mTOR integra i segnali di nutrienti e fattori di crescita

mTOR

Glucosio

ATP

AminoacidiTSC2

TSC1

AMPK

Glucosio

ATP

Segnale di crescita

PI3K

Akt

LAT

GLUT 1

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

Sintesi proteica

Angiogenesi

BioenergiaProliferazione cellulare

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 Circulating Bone Marrow–Derived cell populations that stimulate or amplify tumor angiogenesis.stimulate or amplify tumor angiogenesis.

Kerbel R, New England Journal Medicine,358: 2039-2049, 2008.

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)

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.

potential to differentiate to endothelial cells and contribute to tumor vasculature

Switching in angiogenesis: the two-faces of microenvironment

Noonan DM, Cancer Metastasis Rev.2007

Macrophages, angiogenesis and cancer

Lee et al. J. Cancer Mol. 2(4): 135-140, 2006

TAM produceHIF-1-2

Attract monocyte that differentiate into TAM

Circulating Bone Marrow–Derived cell populations that Circulating Bone Marrow–Derived cell populations that stimulate or amplify tumor angiogenesis.stimulate or amplify tumor angiogenesis.

Kerbel R, New England Journal Medicine,358: 2039-2049, 2008.

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)

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.

potential to differentiate to endothelial cells and contribute to tumor vasculature

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).

The experiments of Lyden and the concept of “premetastatic niche”

Kaplan, R. N. et al. Nature 438, 820–827 (2005).Steeg P. Nature 438, Editorial 750-751 (2005).

BM cells arrive to sites of future metastasis prior to

tumor cells

0

20

40

60

80

100

120

140

Day 8 Day 12 Day 14 Day 16 Day 24

cells

/100

x fi

eld

VEGFR1+

VEGFR2+

Arrival of VEGFR1+ BM and VEGFR2+ Endothelial Cells

Courtesy of David Lyden

Accelerated Metastasis after Short-Term Treatmentwith a Potent Inhibitor of Tumor Angiogenesis

Lobos JBL… Kerbel, Cancer Cell 15, 232–239, March 2009

Accelerated experimental metastasis and decreased survival after short-term sunitinib

treatment before and after Intravenous Tumor Inoculation 231/LM2-4LUC+

No treatmentRx before implantation

Rx after implantation

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.

Jain R, Science 2005; 307: 58

Endothelial cells “sense” O2

and have reduced oxygen consume.

Fre

quen

cy A

mon

g G

FP

+ C

ells

CD11b+

Gr1+

CD11b+Gr1+

0

10

20

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60

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90

100

Anti-VEGF Control

TIB6 B16F1 EL4 LLC TIB6 B16F1 EL4 LLC

* * * + + +* * *

+ + +

0

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2500

1 4 7 10 13 17 21

Legend Primed Mice

B16F1

Cell Line

B16F1

B16F1

B16F1

B16F1

EL4

LLC

None

LLC

EL4

BMMNCs

CD11b+Gr1+

CD11b+Gr1+

CD11b-Gr1-

CD11b-Gr1-

+

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*

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+

+

1000

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Mea

n T

umor

Vol

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(mm

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Reduced responsiveness to anti-VEGF-A therapy following co-injection of CD11b+Gr1+ (but not CD11b-Gr1-) cells from VEGF-independent tumors

Shojaei et al. Nature Biotechnol., 8:911-20, 2007

Refractoriness to anti-VEGF-A is not mediated by the VEGFR-1 specific ligands VEGF-B or PlGF

Anti-Bv8 antibodies suppress tumor angiogenesis and growth

Anti-VEGF Anti-Bv8Control

HM7 tumor

Shojaei et al., Nature 450:825-831, 2007

Additive effects of anti-VEGF and anti-Bv8 in treating established tumors

0

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12 15 18 21 24 27 31 34 37 41 44 47 51 54

Days

ControlAnti-Bv8Anti-VEGFCombination

* * * * * **

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umou

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olum

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m3)

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1200

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7 11 14 17 21 24 31 34 37 41

ControlAnti-Bv8Anti-VEGFCombination

Days

** * *

* *

Mea

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