New perspectives in Tumor Angiogenesis

Institute for Cancer Research and Treatment New perspectives in Tumor Angiogenesis Round Table 2 Italy-Russia@Dubna December 22th, 2010 Federico Bussolino, M.D., Ph.D. Scientific Director Fondazione Piemontese per la Ricerca sul Cancro-Onlus - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

Institute for Cancer Research and Treatment The features and the Mission of the Institute An example of research: tumor angiogenesis Fondazione Piemontese per la Ricerca sul Cancro-Onlus - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

The History 1986: The Fondazione Piemontese per la Ricerca sul Cancro was funded and signed agreements with the University of Torino and the Public Health Ministry 1998: IRCC was completed and the Research activities started 2000: Clinical activities started 2011: Starting of a new building dedicated to basic and translational research activities: the Interdisciplinary Cancer Center Fondazione Piemontese per la Ricerca e la Cura sul Cancro - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

Institute for Cancer Research and Treatment Basic and translation research Clinical activities Molecular and cellular basis of metastatic process Gastro-intestintal cancers Breast cancers Molecular and cllular basis of ersonalized medicine Sarcomas Melanomas Fondazione Piemontese per la Ricerca e la Cura sul Cancro - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

Institute for Cancer Research and Treatment Area: 2500 mq People: 130 Budget 2010: 8800 KEuro Research Area Core facilities: Mouse House: (breeding colonies, stock and experimental mice, genetically modified mice, BSL3 activities): 12000 mice Genomic platform (Illumina platform, real time PRC, Genome Sequencer FLX system, 454 massive DNA sequencing, Fish, ISH, shrna libraries) Microscopy (confocal microscoper, FRET, FRAP, videolapse microscopy, analysis, BD pathway for high throughput screening) FACS sorter (DAKO MoFlo High Speed sorter) Mouse pathology Fondazione Piemontese per la Ricerca e la Cura sul Cancro - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

Institute for Cancer Research and Treatment PhD School: Complex Systems in Medicine and Life Sciences http://dott-scmsv.campusnet.unito.it/cgi-bin/home.pl PhD School: Molecular Medicine http://dott-mm.campusnet.unito.it/cgi-bin/home.pl 3 SME: Horizon Discovery Apavadis Biotechnologies Creabilis Therapeutics Fondazione Piemontese per la Ricerca e la Cura sul Cancro - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

Institute for Cancer Research and Treatment The Mission of Interdisciplinary Cancer Center (ICC) With the completion of the human genome we understood that life is based on dynamic molecular networks rather than on a direct connection between genotype and phenotype. Indeed ICC is interested in is understanding the biological networks supporting the metastatic spreading. Quantitative experimental data obtained at different spatial scales are integrated with the ultimate goal of being able to describe the features of metastasis network and to predict its behaviour. This thrust is thoroughly integrated with the clinical research programs already in place at the Center to build up methods and strategies for a predictive oncology allowing earlier diagnoses and tailored therapies and to build up from bed to bench projects. Fondazione Piemontese per la Ricerca e la Cura sul Cancro - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

Core: Cancer Genetics Cell Biology Animal Models Imputs and Tools from Hard sciences

Cancer - Microenvironment Ping-Pong

How does Cancer - Microenvironment Ping-Pong Influence therapy?

Institute for Cancer Research and Treatment The features and the Mission of the Institute An example of research: tumor angiogenesis Fondazione Piemontese per la Ricerca sul Cancro-Onlus - Fondazione Piemontese per l Oncologia - Università degli Studi di Torino

ANGIOGENESIS MILESTONES 1971: The beginning Isolation of a tumor factor responsible for angiogenesis Folkman et al J Exp Med. 133:275, 1971 1983: VEGF is isolated Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid Senger et al Science 219: 282, 1983 2003: Doubts and hopes Despite the striking effects of several of these antiangiogenic agents on tumor xenografts in mice none has demonstrated efficacy in phase III clinical trials G. Semenza Annu. Rev Med. 54:17, 2003 2004: Clinical Trial Phase III Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. Hurvitz et al N Engl J Med. 2004 350:2335, 2004 2010: The current use Anti-angiogenic regimens are extensively used, but their efficacy is far from the preclinical results

THE VASCULAR UNIT

Free angioblasts develop in the mesoderm Vasculogenesis: Free angioblasts assemble into cords Angioblasts differentiate into endothelium and form tubes assemble into cords Primary plexus Angiogenesis Intususception Sprouting angiogenesis Remodeling and maturation Recruitment of mural cells

AUTACOIDS SHEAR STRESS ANGIOPOIETINS PDGF, TGFβ, Νotch SEMAPHORINS Netrins, Slit MATRIX INTERACTIONS

PARALLEL BETWEEN GENETIC ALTERATIONS DURING PROGRESSION OF COLONRECTAL CARCINOMA AND POSTULATED CHANGES IN TUMOR ANGIOGENESIS PROFILE APC 5qLO K-RAS 18 p LOH 17p LOH OTHERS DCC p53 NORMAL EPITHELIUM HYPERPLASTIC EPITHELIUM EARLY ADENOMA INTERMEDIATE ADENOMA LATE ADENOMA CARCINOMA METASTASIS Haemorrhage? VEGF-A? VEGF-A VEGFR-2 TSP-1

Mechanisms of Tumor Angiogenesis Sprouting Angiogenesis Cooptive Angiogenesis Vasculogenesis (Endothelial Precursors Mobilized from BM) VEGF/SDF-1 mediated mobilization

Too much hypoxia... by aggressive anti- VEGF regimen Ebos et al. Accelerated Metastasis after Short-Term Treatment with a Potent Inhibitor of Tumor Angiogenesis. Cancer Cell 15:232, 2009 Paez-Ribes et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasione and distant metastasis. Cancer Cell 15:220,2009

AUTACOIDS SHEAR STRESS ANGIOPOIETINS PDGF, TGFβ, Νotch SEMAPHORINS Netrins, Slit MATRIX INTERACTIONS

VASCULAR GUIDANCE AND TISSUE ARCHITECTURE CAPILLARY DISTRIBUTION ACCORDING TO O 2 DIFFUSION

Angiogenic remodeling Primary capillary plexus Mature vascular tree Extracellular matrix (ECM)

Andreas Vesalius (1514-1564) B. Anderson, 2002 Green:vessels Red: nerves

AXON GUIDANCE REPULSION ACTRACTION

Class 3 semaphorin inhibits integrin functions and affects vascular remodeling % o f W o w -1 p o s it iv e c e lls 9 8 7 6 5 4 3 2 1 0 Control VEGF-A VEGF-A+ Sema3A RCAS-GFP 35 Endothelial cell haptotaxis Active β1 integrins No. of migrated cells 30 25 20 15 10 5 Control Sema 3A RCAS- Npn-1 Total β1 integrins 0 0 1 2 5 10 Fibronectin (µg/ml) RCAS- PlxA Serini, 2003

SEMA 3A inhibits integrin function by switching-off R-Ras Talin PIP2 Talin GAP domain Rnd Rac-GTP Cytoskeleton GEF molecule Rac-GDP Toyofuku,2005; Serini, 2005

Multistage tumorigenesis in RIP-Tag transgenic mice Normal stage (onc+) Hyperplastic/ Dysplastic stage Angiogenic stage Tumor stage Multistage tumorigenesis in HPV/E 2 transgenic mice N/E2 CIN-1/2 CIN-3 SCC S E E S E E S E T T

Sema3 expression during stages of tumor progression in RipTag2 and in in K14-HPV16/E2 mice Relative quantification (compared to N) Relative quantification (compared to N/E 2 ) 10 H 8 6 4 2 0-2 -4-6 -8-10 Sema 8 6 4 2 0-2 -4-6 Sema 3A 3B 3C 3D 3E 3F 3A 3B 3C 3D 3E 3F A T CIN1-2 CIn3 SCC Sema3A and sema3f are strongly up-regulated in the early stages of tumor angiogenesis and completely switched-off in invasive tumor Maione et al, 2009

Sema3A expression during tumor progression in RipTag2 mice N A T A N T Sema3A N A T Meca32 A A N T Sema3A Meca32 DAPI 630x 400x 400x

Adeno-associated virus (AVV) 8 as a new gene delivery tool for pancreatic cells p5 p19 p46 wtaav polya ITR Rep Cap ITR raav lacz or GFP ITR CMV Promoter lacz or GFP polya ITR pancreas Control AAV8-LacZ

Tumor burden (mm 3 ) n=12 70 Effect of AAV8-Sema3A treatment on tumor growth and vascular density in a regression trial (12-16 weeks) in tumor-bearing RipTag2 160 140 120 100 80 60 40 20 0 * p<0,01 12 wk 16 wk 16 wk T 0 LacZ Sema3A CD31 Control 200X CD31 AAV8-Sema3A 400x 200X % reduction vascular density 60 50 40 30 20 10 CD31 AAV8-Sema3A Exocrine pancreas 200x 0 AAV8-LacZ AAV8-Sema3A Sugen Maione et al, 2008

NORMALIZATION

The landmarks of vascular normalization Improvement of vessel shapes Vessel pruning Interstitial pressure reduction Improvment of oxygen delivery Pericytes recruitment Improvment of drug delivery

Sema3A replacement improves vascular shape

AAV8-Sema3A treatment increases pericyte coverage Control 400x AAV8-Sema3A Meca32 + ΝG2 400x 400x Area density (%) 80 70 60 50 40 30 20 * p<0.01 ** p<0.001 Meca32 NG2 10 0 Control tumors AAV8-Sema3A treated tumors

Sema3A replacement improves Oxygen delivery Low Oxygen Low Oxygen Normal Oxygen

AAV8-sema3A treatment blocks tumor invasion Control RipTag tumors Ac AAV8-sema3A H&E T Ac T Ac Ac Collagen I T T T Ac T

AAV8-sema3A treatment blocks tumor invasion 90 80 70 IT IC1 IC2 % total tumor per animal 60 50 40 30 20 10 0 Control Sunitinib AAV8- Sema3A IT= encapsulated islet tumor IC1= microinvasive tumor IC2= fully invasive tumor

Treatment with Sunitinib, but not with AAV8-Sema3A induces liver metastases in Rip-Tag2 Incidence of liver metastasis 50 40 30 20 10 0 Control Sunitinib AAV8- Sema3A

Exogenous Sema3A