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 2003 1st negative Phase 3 Bev trial reported in MBC 2005-2006 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

The lymphatic “immuno” vascular system 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. 4

Binding del VEGF ai recettori VEGFR-3/Flt-4 VEGFR-1/Flt-1 VEGFR-2/KDR ANGIOGENESI 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 Proliferation Adapted from Graff J, et al. Cancer Res. 2005;65:7462-7469 8

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

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]

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.

VEGF Polymorphism in breast cancer

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

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;15:7471-7478

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 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;116:1767-1775. Motoshima et al. J Physiol. 2006;574:63-71. Edinger and Thompson. Mol Biol Cell. 2002;13:2276-2288. Sintesi proteica Proliferazione cellulare Bioenergia Angiogenesi 22 22

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 TAM produce HIF-1-2 Attract monocyte that differentiate into TAM 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 ‘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 750-751 (2005).

Arrival of VEGFR1+ BM and VEGFR2+ Endothelial Cells 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

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

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

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 450:825-831, 2007