VEGF and Cancer (Medical Intelligence Unit)

MEDICAL INTELLIGENCE UNIT

Judith H. Harmey

VEGF and Cancer

MEDICAL INTELLIGENCE UNIT

VEGF and Cancer Judith H. Harmey, Ph.D. Department of Surgery Royal College of Surgeons in Ireland Education and Research Centre Beaumont Hospital Dublin, Ireland

LANDES BIOSCIENCE / EUREKAH.COM GEORGETOWN, TEXAS U.S.A.

KLUWER ACADEMIC / PLENUM PUBLISHERS NEW YORK, NEW YORK U.S.A.

VEGF AND CANCER Medical Intelligence Unit Landes Bioscience / Eurekah.com Kluwer Academic / Plenum Publishers Copyright ©2004 Eurekah.com and Kluwer Academic / Plenum Publishers All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system; for exclusive use by the Purchaser of the work. Printed in the U.S.A. Kluwer Academic / Plenum Publishers, 233 Spring Street, New York, New York, U.S.A. 10013 http://www.wkap.nl/ Please address all inquiries to the Publishers: Landes Bioscience / Eurekah.com, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512.863.7762; Fax: 512.863.0081 http://www.eurekah.com http://www.landesbioscience.com ISBN: 0-306-47988-5 VEGF and Cancer, edited by Judith H. Harmey, Landes / Kluwer dual imprint / Landes series: Medical Intelligence Unit While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Library of Congress Cataloging-in-Publication Data VEGF and cancer / [edited by] Judith H. Harmey. p. ; cm. -- (Medical intelligence unit) Includes bibliographical references and index. ISBN 0-306-47988-5 1. Vascular endothelial growth factors--Pathophysiology. 2. Cancer--Pathophysiology. 3. Neovascularization-Regulation. 4. Tumors--Blood-vessels--Growth. I. Harmey, Judith H. II. Series: Medical intelligence unit (Unnumbered : 2003). [DNLM: 1. Neoplasms--physiopathology. 2. Neoplasms--etiology. 3. Tumor Markers, Biological. 4. Vascular Endothelial Growth Factors--adverse effects. 5. Vascular Endothelial Growth Factors--physiology. QZ 200 V422 2004] RC254.6.V44 2004 616.99'4071--dc22 2004005641

CONTENTS Preface .................................................................................................. xi Abbreviations ...................................................................................... xiii 1. VEGF and Its Receptors ......................................................................... 1 Napoleone Ferrara Activities of VEGF ................................................................................ 1 VEGF Isoforms ..................................................................................... 2 Regulation of VEGF Gene Expression ................................................... 2 The VEGF Receptors ............................................................................ 3 Role of VEGF in Physiological Angiogenesis ......................................... 5 Role of VEGF in Pathologic Conditions ............................................... 6 Therapeutic Implications and Perspectives ............................................ 7 2. Hypoxic Regulation of VEGF .............................................................. 12 Nina S. Levy, Ilana Goldberg-Cohen and Andrew P. Levy Transcriptional Regulation of VEGF ................................................... 12 Post Transcriptional Regulation of VEGF ........................................... 13 Abnormalities in the Hypoxic Regulation of VEGF ............................ 16 3. Molecular Mechanisms of VEGF-Induced Angiogenesis ...................... 19 Sandra Donnini, Marina Ziche and Lucia Morbidelli Angiogenesis ........................................................................................ 19 The Role of Vascular Endothelial Growth Factor in Angiogenesis ....... 19 The VEGF Signal Cascade in Angiogenesis ......................................... 21 4. Crosstalk between VEGF and Bcl-2 in Tumor Progression and Angiogenesis .................................................................................. 26 Donatella Del Bufalo, Daniela Trisciuoglio and Michele Milella Angiogenic Potential of Tumor Cells Overexpressing Bcl-2 ................. 26 Role of Bcl-2 in VEGF-Promoted Survival of Tumor Cells ................. 29 Role of Bcl-2 in Endothelial Cells ........................................................ 29 Clinical Relevance of VEGF and Bcl-2 Expression .............................. 31 5. Vascular Endothelial Growth Factor in Breast Cancer .......................... 40 Tilmann Lantzsch and Lukas Hefler VEGF and Tumor Growth ................................................................. 41 Serum VEGF and Breast Cancer ......................................................... 42 VEGF and Clinical Consequences ....................................................... 43 Future Strategies in Breast Cancer ....................................................... 44 6. VEGF and Tumor Progression in Human Melanoma .......................... 48 Domenico Ribatti, Angelo Vacca and Franco Dammacco VEGF in Tumor Angiogenesis ............................................................ 48 Angiogenesis and Human Melanoma .................................................. 49 VEGF in Human Melanoma ............................................................... 50

7. VEGF in Esophageal Cancer ................................................................ 54 Axel Kleespies, Markus Guba, Karl-Walter Jauch and Christiane J. Bruns VEGF in Squamous Cell Carcinoma of the Esophagus ....................... 54 VEGF in Barrett’s Disease and Adenocarcinoma of the Esophagus ............................................................................. 55 Circulating VEGF Levels in Esophageal Carcinoma ............................ 56 VEGF and MVD during Neoadjuvant Treatment of Esophageal Carcinoma ................................................................ 57 VEGF-C and Lymphangiogenesis in Esophageal Carcinoma ............... 59 Anti-VEGF Treatment of Esophageal Carcinoma ............................... 60 8. VEGF in Colorectal Cancer .................................................................. 64 Markus Guba, Hendrik Seeliger, Karl-Walter Jauch and Christiane J. Bruns Colon Cancer ...................................................................................... 64 VEGF in Colorectal Cancer Progression .............................................. 64 Prognostic Value of VEGF in Colorectal Cancer Patients .................... 66 The Role of VEGF in Metastasis of Colorectal Cancer ........................ 66 Anti-Angiogenic Therapy by Interference with the VEGF Pathway in Colorectal Cancer ......................................................... 68 9. Vascular Endothelial Growth Factor in Malignant Disease of the Central Nervous System ............................................................. 72 David Stefanik VEGF in the Normal Brain ................................................................. 72 VEGF Is Upregulated in Malignant Disease of the CNS ..................... 72 Angiogenesis in Glioma ....................................................................... 74 Factors Influencing VEGF Production ................................................ 76 C6 Glioma Is an Excellent Model for the Study of High Grade Human Glioma with Regard to VEGF ............................................ 76 VEGF Is Responsible for the Virulent Nature of High Grade Gliomas ................................................................................ 77 Interrupting VEGF-Receptor Signaling Inhibits Glioma Growth in Preclinical Models .......................................................... 77 Current Therapy of Glioma: VEGF Contributes to Treatment Failure ....................................................................... 78 10. VEGF in Hematopoietic Malignancy ................................................... 83 Philip T. Murphy and John Quinn The VEGF/VEGF Receptor Pathway .................................................. 84 Hematopoietic and Endothelial Cells Share a Common Hematopoietic/Endothelial Progenitor Cell ..................................... 84 VEGF Bone Marrow Interactions in Hematological Malignancy ......... 85 VEGF and Angiogenesis in Acute Leukemia and Myelodysplasia ........ 88 VEGF and Angiogenesis in Myeloproliferative Disorders .................... 90

VEGF and Angiogenesis in B-Cell Chronic Lymphocytic Leukemia ......................................................................................... 92 VEGF and Angiogenesis in Lymphomas ............................................. 93 VEGF and Angiogenesis in Myeloma .................................................. 94 VEGF Signalling Pathways As a Therapeutic Target in Hematological Malignancies ........................................................ 95 11. Targeting VEGF in Pancreatic Cancer ............................................... 107 Cheryl H. Baker, Carmen C. Solorzano and Isaiah J. Fidler Cancer Metastasis .............................................................................. 107 Tumor Angiogenesis .......................................................................... 107 Regulation of Angiogenesis by the Microenvironment ....................... 109 VEGF/VPF: A Pro-Angiogenic Molecule .......................................... 109 Regulation of VEGF/VPF Expression in Tumors .............................. 110 Development of a Human Pancreatic Adenocarcinoma Model .......... 110 Anti-VEGF Therapy in Pancreatic Cancer ........................................ 111 Anti-Angiogenic Therapy: Clinical Implications ................................ 112 12. Effects of Fibrinogen and Associated Peptide Fragments on the Activation of Human Endothelial Cells by VEGF in Vitro ............................................................................... 117 Carolyn A. Staton, Nicola J. Brown and Claire E. Lewis Fibrinogen and Fibrin Formation ...................................................... 118 Fibrinolysis ........................................................................................ 118 Effects of Fibrinogen on Endothelial Cell Activation ......................... 118 Fibrin, VEGF and Angiogenic Mechanisms ...................................... 124 Stimulation of VEGF Induced Angiogenesis by Fibrin E-Fragment ................................................................................... 126 Fibrinogen E-Fragment Inhibits VEGF Activation of Endothelial Cells ....................................................................... 129 The Effects of Other Fibrinogen/Fibrin Related Fragment on VEGF-Activation of Endothelial Cells ...................................... 130 13. Vascular Endothelial Growth Factor (VEGF) and Its Role in Non-Endothelial Cells: Autocrine Signalling by VEGF .................. 133 Angela M. Duffy, David J. Bouchier-Hayes and Judith H. Harmey VEGF in the Cardiovascular System .................................................. 133 VEGF and the Central Nervous System (CNS) ................................. 134 VEGF and Its Role in Bone ............................................................... 135 VEGF in Hematopoietic Cells and Hematological Malignancies ....... 135 VEGF Signalling in Hematopoietic Cells .......................................... 136 Evidence for VEGF Autocrine Signalling in Solid Tumors ................ 136 Autocrine VEGF Signalling in Breast Cancer .................................... 137 VEGF Stimulates Breast Cancer Invasion .......................................... 137 VEGF Signalling in Tumor Cells ...................................................... 138 Anti-Angiogenic Therapy .................................................................. 139

14. Vascular Endothelial Growth Factor C and Vascular Endothelial Growth Factor D: Biology, Functions and Role in Cancer ................. 145 Sarah E. Duff and Gordon C. Jayson Molecular Biology of VEGF-C and VEGF-D ................................... 145 VEGF-C and VEGF-D Signalling and Function ............................... 149 VEGF-C and VEGF-D in Human Malignancy ................................. 152 Potential Therapeutic Roles ............................................................... 156 Index .................................................................................................. 163

EDITOR Judith H. Harmey, Ph.D. Department of Surgery Royal College of Surgeons in Ireland Education and Research Centre Beaumont Hospital Dublin, Ireland E-mail: [email protected] Chapter 13

CONTRIBUTORS Cheryl H. Baker Department of Surgery Children’s Hospital Boston, Massachusetts, U.S.A. Email: [email protected] Chapter 11

Franco Dammacco Department of Biomedical Sciences and Human Oncology University of Bari Medical School Bari, Italy Email: [email protected] Chapter 6

David J. Bouchier-Hayes Department of Surgery Royal College of Surgeons in Ireland Education and Research Centre Beaumont Hospital Dublin, Ireland Email: [email protected]

Donatella Del Bufalo Experimental Chemotherapy Laboratory Regina Elena Cancer Institute Rome, Italy Email: [email protected] Chapter 4

Chapter 13

Nicola J. Brown Microcirculation Research Unit University of Sheffield Medical School Sheffield, U.K. Email: [email protected]

Sandra Donnini Department of Molecular Biology University of Siena Siena, Italy Email: [email protected] Chapter 3

Chapter 12

Christiane J. Bruns Department of Surgery University-Hospital Grosshadern Ludwig-Maximilians-University Munich, Germany Email: [email protected] Chapters 7, 8

Sarah E. Duff Department of Surgery Christie Hospital NHS Trust Manchester, U.K. Email: [email protected] Chapter 14

Angela M. Duffy Department of Surgery Royal College of Surgeons in Ireland Education and Research Centre Beaumont Hospital Dublin, Ireland Email: [email protected]

Karl-Walter Jauch Department of Surgery University-Hospital Grosshadern Ludwig-Maximilians-University Munich, Germany Email: [email protected] Chapters 7, 8

Chapter 13

Napoleone Ferrara Department of Molecular Oncology Genentech, Inc. South San Francisco, California, U.S.A. Email: [email protected]

Gordon C. Jayson Department of Medical Oncology Christie Hospital NHS Trust Manchester, U.K. Email: [email protected]

Chapter 1

Chapter 14

Isaiah J. Fidler Department of Cancer Biology University of Texas MD Anderson Cancer Center Houston, Texas, U.S.A. Email: [email protected]

Axel Kleespies Department of Surgery University-Hospital Grosshadern Ludwig-Maximilians-University Munich, Germany Email: [email protected]

Chapter 11

Chapter 7

Ilana Goldberg-Cohen Bruce Rappaport Faculty of Medicine and Rappaport Family Institute for Research in the Medical Sciences Technion-Israel Institute of Technology Haifa, Israel Email: [email protected]

Tilmann Lantzsch Department of Obstetrics and Gynecology St. Elisabeth and St. Barbara Hospital Halle/Saale, Germany Email: [email protected]

Chapter 2

Chapter 5

Markus Guba Department of Surgery University-Hospital Grosshadern Ludwig-Maximilians-University Munich, Germany Email: [email protected]

Andrew P. Levy Bruce Rappaport Faculty of Medicine and Rappaport Family Institute for Research in the Medical Sciences Technion-Israel Institute of Technology Haifa, Israel Email: [email protected]

Chapters 7, 8

Chapter 2

Lukas Hefler Department of Obstetrics and Gynecology University of Vienna Vienna, Austria Email: [email protected] Chapter 5

Angelo Vacca Department of Biomedical Sciences and Human Oncology University of Bari Medical School Bari, Italy Email: [email protected] Chapter 6

Marina Ziche Department of Molecular Biology University of Siena Siena, Italy Email: [email protected] Chapter 3

PREFACE

I

t is now firmly established that the growth of new blood vessels— angiogenesis—is critical to both the growth and metastasis of solid tumors. In addition to solid tumors, angiogenesis is also a feature of hematological malignancies. Angiogenesis is regulated by the overall balance of angiogenesis promoters and angiogenesis inhibitors. Vascular endothelial growth factor, VEGF, also known as vascular permeability factor, VPF, is one of the most potent and studied of the angiogenic factors. VEGF-A, usually referred to as VEGF, is one of a gene family of growth factors which includes VEGF A-E and placental growth factor (PlGF). The main focus of this book is VEGF-A, although the other members are discussed, particularly VEGF-C and D. VEGF exerts its angiogenic effects by interacting with specific receptors on endothelial cells stimulating their growth and differentiation into blood vessels. VEGF-C and D interact with receptors on the lymphatic endothelium to stimulate lymphangiogenesis which may be important in lymphatic metastasis. This book focuses on the role and regulation of VEGF in cancer. Chapters 1-4 focus on the molecular features of VEGF and VEGF signalling pathways in the cell. Chapter 1 focuses on the biology of VEGF and its receptors and describes its role in physiological and pathological angiogenesis, as well as the therapeutic potential of VEGF blockade. Hypoxia, low oxygen tension, is a feature of solid tumors and Chapter 2 examines the critical role of hypoxia in regulating VEGF expression. Chapters 3 and 4 describe in detail the molecular pathways involved in VEGF signal transduction. Chapters 5-9 discuss the role of VEGF in a range of solid tumors—breast cancer, melanoma, esophageal cancer, colorectal cancer and tumors of the central nervous system, respectively—and Chapter 10 focuses on the role of VEGF in hematopoietic malignancies. In Chapter 11, the efficacy of blocking VEGF signalling in pancreatic cancer is discussed while Chapter 12 discusses the interaction between fibrinogen and peptides derived from it, and VEGF. One particular fragment, fibrinogen E, is shown to inhibit the angiogenic activity of VEGF whereas others promote VEGF-induced angiogenesis. Chapter 13 explores the role of VEGF in non-endothelial cells, both normal host cells and tumor cells, and emerging evidence for the existence of autocrine signalling pathways as well as tumor cell-endothelial cell paracrine signalling. Chapter 14 discusses the role of the lymphangiogenic members of the VEGF family, VEGF-C and VEGF-D, in cancer and their potential as prognostic markers and therapeutic targets.

This book is intended to provide a comprehensive understanding of VEGF and VEGF signalling pathways in tumors and an overview of its role in a variety of different tumor types. Furthermore, the enormous clinical potential of blocking VEGF and/or VEGF signalling using a variety of approaches is discussed in many chapters. Strategies to block the angiogenic activity of VEGF include monoclonal antibodies, tyrosine kinase inhibitors targeting the VEGF receptors, antisense oligonucleotides and soluble VEGF receptor fragments which bind VEGF but do not transduce a signal, and molecules targeting elements of the VEGF signal transduction pathway such as rapamycin. Some of these approaches are already in clinical trials (Table 1, Chapter 8) and have shown clinical promise particularly when used in combination with conventional chemotherapy or radiotherapy. It should be a useful reference text for both established scientists and those new to the area. The chapters dealing with VEGF in specific tumor types (Chapters 5-11) may be of particular interest to clinicians. For readers wishing to investigate a particular topic in more detail, each chapter gives an extensive and up to date list of references—both original articles and reviews. I am grateful to all the graduate students, post-doctoral fellows and research assistants that have passed through my lab and taught me as much as I taught them. The support, patience and encouragement of my husband James and my parents, Matt and Rose, throughout my career has been essential. Prof. David Bouchier-Hayes and Dr. Isaiah (Josh) Fidler have both been inspirational role models. Finally, and most importantly, I thank all the authors for their time and excellent contributions to this book. Judith H. Harmey, Ph.D.

ABBREVIATIONS 4E-BP1 5-FU Ab AC aFGF Akt ALL AMD AML AP-1 or -2 APAF-1 APC APL ATL ATRA AU rich element B2MG BAEC bFGF BM BMA BMMNC bp C CAD CAM CBP cDNA CEC c-fos CFU CGNs CL CLL CM CML CMML CNS COX-2 CRT CSF

4E-binding protein 1 5-Fluorouracil antibodies adenocarcinoma acidic fibroblast growth factor Protein Kinase B/PKB acute lymphoblastic leukemia age-related macular degeneration acute myeloid leukemia activator protein-1 or -2 apoptotic protease-activating factor-1 adenomatous polyposis coli acute promyelocytic leukemia adult T cell leukemia all trans retinoic acid adenylate-uridine rich element beta 2 microglobulin bovine aortic endothelial cells basic fibroblast growth factor bone marrow bone marrow angiogenesis bone marrow mononuclear cells base pairs carboxy coronary artery disease chick embyro (or chicken) chorioallantoic membrane camp response element copy DNA circulating endothelial cells a member of the family of intermediate early genes colony forming unit cerebellar granule neurons corpus luteum chronic lymphocytic leukemia conditioned medium chronic myeloid leukemia chronic myelomonocytic leukemia central nervous system cyclooxygenase 2 chemo-radiotherapy colony stimulating factors

CT CXCR4 DNA E EC ECM EGF EGFR EG-VEGF eIF-4E ELAV ELISA eNOS EP2 ER ERK-1 ERK-2 ET FAB FAK FAP FDP FGF-2 Fgn FgnD FgnE FIGF FKBP Flk-1 Flt-1 Flt-4 FnE FpA FpB FTI GAP G-CSF GERD GFR GM-CSF G-protein Gy HD HDMEC HE

computerized tomography chemokine receptor 4 deoxyribonucleic acid embryonic day endothelial cells extracellular matrix epidermal growth factor epidermal growth factor receptor endocrine-gland derived VEGF eukaryotic translation initiation factor 4E embryonic lethal abnormal vision enzyme-linked immunosorbent assay endothelial nitric oxide synthase prostaglandin receptor subtype 2 estrogen receptor extracellular-regulated kinase-1 extracellular-regulated kinase-2 essential thrombocythemia French American British focal adhesion kinase familial adenomatous polyposis fibrin degradation products fibroblast growth factor 2 (bFGF) fibrinogen fibrinogen D-fragment fibrinogen E-fragment c-fos induced growth factor FK-binding protein fetal liver kinase 1, the murine homologue of human KDR (VEGFR-2) fms-like tyrosine kinase 1 (VEGFR-1) fms-like tyrosine kinase 4 (VEGFR-3) fibrin E-fragment fibrinopeptide A fibrinopeptide B farnesyl transferase inhibitors GTPase activating protein granulocyte-CSF gastro esophageal reflux disease growth factor reduced granulocyte monocyte colony stimulating factor guanine nucleotide-binding protein gray Hodgkin’s disease human dermal microvascular endothelial cells hematopoietic/endothelial

HGF HGF/SF HIF HIF-1 HIF-1 α HNSCC HPC HRE HSCs HSP90 HUVEC IAP ICAM-1 IFN IFP Ig IGF-1 IGF-1R IHC IKK IL IL-1β IL-6 IL-8 ImiDs iNOS IRES ISH JUNK Kb Kd KDR KGF Ki-67 LDH LSEC LVD LYVE-1 M+ mAb MAPK M-CSF MDS MGUS MK MM

hepatocyte growth factor hepatocyte growth factor/scatter factor hypoxia inducible factor hypoxia inducible factor-1 hypoxia inducible factor 1 alpha head and neck squamous cell cancer hematopoietic progenitor cells hypoxia response element hematopoietic stem cells heat shock protein 90 human umbilical vascular endothelial cells inhibitor of apoptosis protein intercellular adhesion molecule-1 interferon interstitial fluid pressure immunoglobulin insulin-like growth factor-1 IGF-1 receptor immunohistochemistry IκB kinase interleukin interleukin-1 beta interleukin-6 interleukin-8 immunomodulatory drugs inducible nitric oxide synthase internal ribosome entry site in situ hybridisation c-Jun N-terminal kinase kilobase pairs dissociation constant kinase insert domain-containing receptor (flk-1, VEGFR-2) keratinocyte growth factor antibody, which reacts with nuclei of actively proliferating cells lactic dehydrogenase liver sinusoidal endothelial cells lymphatic vessel density lymphatic vessel hyaluronan receptor-1 presence of distant metastases (TNM classification) monoclonal antibody mitogen activated protein kinase monocyte-CSF myelodysplastic syndromes monoclonal gammopathy of undetermined significance megakaryocytes multiple myeloma

MMM MMP MMP-9 MPD mRNA mTOR MTT MVD N NA NF-1 NFκB NHL NO NOD-SCID NP-1 NS NSCLC OA p53 PB PC PCL PCLI PCR PD-ECGF PDGF PDGF-BB PI3-K PKC PLA PLCγ PlGF pM PMA pN pN0 pO2 pT PTMM PV P-VEGF Raf-1 RGD rhu mAb

myelofibrosis with myeloid metaplasia matrix metalloproteinase matrix metalloproteinase-9 myeloproliferative disorders messenger RNA mammalian target of rapamycin 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide microvessel density amino not assessed nuclear factor-1 nuclear transcription factor kappa B non-Hodgkins-lymphoma nitric oxide non-obese diabetic-severe combined immunodeficient neuropilin-1 not specified non-small cell lung cancer osteoarthritis tumor suppressor protein peripheral blood plasma cell plasma cell leukemia plasma cell labelling index polymerase chain reaction platelet derived endothelial cell growth factor platelet derived growth factor platelet derived growth factor-BB phosphatidylinositol 3’-kinase protein kinase C phospholipase A phospholipase Cγ placental growth factor picomolar phorbol myristate 12,13 acetate node status (TNM classification) node negative (TNM classification) oxygen tension extension of primary tumor (TNM classification) postthrombocythemic myeloid metaplasia polycythemia vera plasma VEGF raf-1 kinase arginine-glycine-aspartic acid humanized monoclonal antibody

RIP RNA RPA R-S RTK RTKI RT-PCR RTQ-RT-PCR SCC SCF SCID SDF-1 sFlt-1 SLL SMC sNP-1 Sp-1 S-VEGF TF TGF TGF-α TGF-β TGF-β1 Tie-2/Tek TNF TNF α TNM Tre-1 TUNEL

rat insulin promoter ribonucleic acid ribonuclease protection assay Reed-Sternberg receptor tyrosine kinase receptor tyrosine kinase inhibitors reverse transcriptase - polymerase chain reaction real time quantitative - RT-PCR squamous cell carcinoma stem cell factor severe combined immunodeficient stromal cell derived factor-1 soluble Flt-1 small lymphocytic lymphoma smooth muscle cells soluble NP-1 specificity protein-1 serum VEGF tissue factor transforming growth factor transforming growth factor alpha transforming growth factor beta transforming growth factor beta 1 tyrosine kinase receptor for angiopoietin tumor necrosis factor tumor necrosis factor alpha tumor classification system thioredoxin terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling UTR untranslated region VE-cadherin vascular endothelial cadherin VEGF vascular endothelial growth factor VEGF/VPF vascular endothelial growth factor/vascular permeability factor VEGF121, VEGF165, VEGF189: VEGF isoforms, splice variants of VEGF gene VEGF-C vascular endothelial growth factor C VEGF-D vascular endothelial growth factor D VEGFR-1 vascular endothelial growth factor receptor-1 (Flt-1) VEGFR-2 vascular endothelial growth factor receptor-2 (Flk-1, KDR) VEGFR-3 vascular endothelial growth factor receptor-3 (Flt-4) VHL von Hippel-Lindau VPF vascular permeability factor VRP VEGF-related protein, otherwise known as VEGF-C WB western blot ZK7 transcription factor, member of the Krüppel family of genes

CHAPTER 1

VEGF and Its Receptors Napoleone Ferrara

Abstract

T

he development of a vascular supply is a highly complex process. Work done over the last decade has elucidated the critical role of vascular endothelial growth factor (VEGF) in the regulation of normal and pathological angiogenesis. The activities of VEGF are mediated by two tyrosine kinase receptors, VEGFR-1 and VEGFR-2. VEGF is required for embryogenesis, skeletal growth and reproductive functions. Furthermore, VEGF is a mediator of angiogenesis associated with tumors and ischemic retinal diseases. Recent data demonstrated that administration of an anti-VEGF monoclonal antibody results in clinical benefit in patients with metastatic colorectal cancer.

Introduction The existence of angiogenic factors was initially postulated on the basis of the strong neovascular response induced by transplanted tumors.1 Subsequently, it was shown that normal tissues are also a source of angiogenic activity. Many molecules have been implicated as positive regulators of angiogenesis including acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-α and -β, hepatocyte growth factor/scatter factor (HGF/SF), tumor necrosis factor (TNF)-α, angiogenin, interleukin (IL)-8, and the angiopoietins.2,3 For over a decade, the role of vascular endothelial growth factor (VEGF) in the regulation of angiogenesis had been object of intense investigation. For a historic overview of the VEGF field, see ref. 4. While recent evidence indicates that new vessel growth and maturation are highly complex and co-ordinated processes, requiring the sequential activation of a series of receptors by numerous ligands (for reviews see refs. 3, 5, 6), VEGF signalling often represents a critical rate-limiting step in physiological angiogenesis. VEGF appears to be also important in pathological angiogenesis, such as that associated with tumor growth.7 VEGF (referred to also as VEGF-A) belongs to a gene family that includes placenta growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D. Additionally, homologues of VEGF have been identified in the genome of the parapoxvirus, Orf virus and shown to have VEGF-like activities.7,8 The main focus of this review is the biology of the prototype member, VEGF-A, a key regulator of blood vessel growth. Importantly, VEGF-C and VEGF-D regulate lymphatic angiogenesis,9 emphasizing the unique role of this gene family in controlling growth and differentiation of multiple anatomic components of the vascular system.

Activities of VEGF VEGF promotes growth of vascular endothelial cells derived from arteries, veins and lymphatics (for review see ref 7). VEGF induces a potent angiogenic response in a variety of in vivo models.10,11 VEGF delivery also induces lymphangiogenesis in mice.12 While endothelial cells are the primary target of VEGF, several studies have also reported mitogenic effects on certain non-endothelial cell types (reviewed in ref. 13). VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

2

VEGF and Cancer

VEGF is a survival factor for endothelial cells, both in vitro and in vivo.14-17 In vitro, VEGF prevents apoptosis induced by serum-starvation. Gerber et al have shown that such activity is mediated by the phosphatidylinositol 3'-kinase (PI3-K)/Akt pathway.15 Also, VEGF induces expression of the anti-apoptotic proteins, Bcl-2 and A1, in endothelial cells.14 In vivo, VEGF’s pro-survival effects are developmentally regulated. VEGF inhibition results in extensive apoptotic changes in the vasculature of neonatal, but not adult mice.18 Furthermore, a marked VEGF-dependence has been demonstrated in endothelial cells of newly formed but not of established vessels within tumors.16,17 VEGF has also effects on bone marrow-derived cells. It promotes monocyte chemotaxis19 and induces colony formation by mature subsets of granulocyte-macrophage progenitor cells.20 VEGF delivery to adult mice inhibited dendritic cell development21 and increased production of B cells and generation of immature myeloid cells.22 Conditional gene-knock out technology has been employed to achieve selective VEGF gene ablation in bone marrow cell isolates and hematopoietic stem cells (HSCs).23 VEGF deficient HSCs and bone marrow mononuclear cells failed to repopulate lethally irradiated hosts, despite the co-administration of a large excess of wild-type cells. These studies also elucidated an internal autocrine loop, not blocked by extracellular inhibitors like antibodies, by which VEGF controls HSC survival during hematopoietic repopulation.23 VEGF is known also as vascular permeability factor (VPF), based on its ability to induce vascular leakage.24,25 It is now well established that such permeability-enhancing activity underlies significant roles of this molecule in inflammation and in other pathological circumstances. VEGF induces an increase in hydraulic conductivity of isolated microvessels and such an effect is mediated by increased calcium influx.26

VEGF Isoforms

The human VEGF-A gene is organized in eight exons, separated by seven introns.27,28 Alternative exon splicing was initially shown to result in the generation of four different isoforms, having respectively 121, 165, 189 and 206 amino acids following signal sequence cleavage (VEGF121, VEGF165, VEGF189, VEGF206).27,28 VEGF165, the predominant isoform, lacks the residues encoded by exon 6, while VEGF121 lacks the residues encoded by exons 6 and 7. Less frequent splice variants have been also reported, such as VEGF145 and VEGF183.8 Native VEGF is a heparin-binding homodimeric glycoprotein of 45 kD.29 The properties of native VEGF closely correspond to those of VEGF165.30 VEGF121 is an acidic polypeptide that does not bind heparin.30 VEGF189 and VEGF206 are highly basic and bind to heparin with high affinity.30 While VEGF121 is a freely diffusible protein, VEGF189 and VEGF206 are almost completely sequestered in the extracellular matrix (ECM). VEGF165 has intermediary properties, as it is secreted, but a significant fraction remains bound to the cell surface and ECM.31 The ECM-bound isoforms may be released in a diffusible form by plasmin cleavage at the COOH terminus, which generates a bioactive fragment.30

Regulation of VEGF Gene Expression Oxygen Tension Oxygen tension (pO2) plays a key role in regulating the expression of a variety of genes. VEGF mRNA expression is induced by exposure to low pO2 in a variety of pathophysiological circumstances.32 It is now well established that hypoxia inducible factor (HIF)-1 is a key mediator of hypoxic responses.33 Recent studies have uncovered the critical role of the product of the von Hippel-Lindau (VHL) tumor suppressor gene in HIF-1-dependent hypoxic responses (for review see ref. 34). The VHL gene is inactivated in patients with von Hippel-Lindau disease, a condition characterized by capillary hemangioblastomas in retina and cerebellum, and in most sporadic clear cell renal carcinomas. Most of the endothelial cell mitogenic activity released by renal cell carcinoma cells expressing a mutant VHL was neutralized by anti-VEGF antibodies.35 One function of the VHL protein is negative regulation of VEGF and other

VEGF and Its Receptors

3

hypoxia-inducible genes.36 HIF-1 was shown to be constitutively activated in VHL-deficient renal cell carcinoma cell lines.37 Other studies demonstrated that one of the functions of VHL is to be part of a ubiquitin ligase complex that targets HIF-1 subunits for proteasomal degradation following covalent attachment of a polyubiquitin chain (for review see ref. 38). Oxygen promotes the hydroxylation of HIF-1 at proline residues, a requirement for the association with VHL. Recently, a family of prolyl hydroxylases were identified as HIF prolyl hydroxylases.39

Growth Factors and Oncogenes Several major growth factors, including epidermal growth factor (EGF), TGF-α, TGF-β, keratinocyte growth factor (KGF), insulin-like growth factor (IGF)-1, FGFs and platelet derived growth factor (PDGF), up-regulate VEGF mRNA expression, suggesting that paracrine or autocrine release of such factors co-operates with local hypoxia in regulating VEGF release in the microenvironment.7,8 Also, inflammatory cytokines such as IL-1α and IL-6 induce expression of VEGF in several cell types, including synovial fibroblasts, in agreement with the hypothesis that VEGF may be a mediator of angiogenesis/permeability in inflammatory disorders.8 Specific transforming events, such as mutations or amplification of the ras gene, lead to VEGF up-regulation.40,41

The VEGF Receptors Initially, VEGF binding sites were identified on the cell surface of vascular endothelial cells, in vitro and in vivo. Subsequently, it became apparent that receptors for VEGF exist also on bone marrow-derived cells.7 VEGF binds two related receptor tyrosine kinases (RTK), VEGFR-1 (Flt-1, fms-like tyrosine kinase) and VEGFR-2 (Flk-1/KDR, Fetal liver kinase 1, murine homologue of human Kinase insert Domain-containing Receptor). Both VEGFR-1 and VEGFR-2 have seven immunoglobulin (Ig) like domains in the extracellular domain, a single transmembrane region and a consensus tyrosine kinase sequence which is interrupted by a kinase-insert domain.42,43 In addition to these RTKs, VEGF interacts with a family of coreceptors, the neuropilins (NP).

VEGFR-1(Flt-1)

VEGFR-1/Flt-1 was the first RTK to be identified as a VEGF receptor a decade ago,44 but its precise function is still the subject of debate. Recent evidence indicates that the conflicting reports may be due, at least in part, to the fact that VEGFR-1 functions and signalling properties can be different depending on the developmental stage of the animal and the particular cell type, for example endothelial versus bone marrow cells. VEGFR-1 expression is upregulated by hypoxia via a HIF-1 dependent mechanism.45 VEGFR-1 binds not only VEGF-A but also placental growth factor (PlGF)46 and VEGF-B,47 which fail to bind VEGFR-2. The binding site for VEGF (and PlGF) has been mapped primarily to the second Ig-like domain.48 Flt-1 reveals weak tyrosine autophosphorylation in response to VEGF.44,49 Park et al initially proposed that VEGFR-1 may be not primarily a receptor transmitting a mitogenic signal, but rather a “decoy” receptor, able to regulate in a negative fashion the activity of VEGF on the vascular endothelium, by preventing VEGF binding to VEGFR-2.46 Thus, the observed potentiation of the action of VEGF by PlGF could be explained, at least in part, by displacement of VEGF from VEGFR-1 binding.46 Recent studies have shown that indeed a synergism exists between VEGF and PlGF in vivo, especially during pathological situations, as evidenced by impaired tumorigenesis and vascular leakage in PlGF-/- mice.50 Gille et al have identified a repressor motif in the juxtamembrane region of VEGFR-1 that impairs PI3-K activation in response to VEGF.51 Gene targeting studies have demonstrated that Flt-1-/- mice die in utero between day 8.5 and 9.5.52 Endothelial cells develop but fail to organize in vascular channels. Excessive proliferation of angioblasts has been reported to be responsible for the lethality, suggesting that, at least during early development, VEGFR-1 is a negative regulator of VEGF

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VEGF and Cancer

action. Furthermore, targeted mutation resulting in a VEGFR-1 lacking the tyrosine kinase (TK) domain, but able to bind VEGF, does not result in lethality or any overt defect in vascular development.53 Recently, VEGFR-1 signaling has been linked to the induction of matrix metalloproteinase 9 (MMP-9) in lung endothelial cells and to the facilitation of lung metastases.54 Recent studies have emphasized the role of VEGFR-1 in hematopoiesis and recruitment of endothelial progenitors. Hattori et al have shown that VEGFR-1 activation by PlGF is able to reconstitute hematopoiesis by recruiting VEGFR-1+ HSC.55 In addition, Gerber et al have shown that VEGFR-1 activation rescues the ability to repopulate in VEGF-/- HSC.23 Furthermore, Luttun et al have shown that PlGF promotes collateral vessel growth in a model of myocardial ischemia through the recruitment of monocytes.56 LeCouter et al provided evidence for a novel function of VEGFR-1 in liver sinusoidal endothelial cells (LSEC). VEGFR-1 activation resulted in the paracrine release of HGF, IL-6 and other hepatotrophic molecules by LSEC, such that hepatocytes were stimulated to proliferate when co-cultured with LSECs.57 Such a mechanism protected the liver from toxic damage, in spite of the inability of the VEGFR-1 agonist to induce LSEC proliferation.

VEGFR-2 (KDR/Flk-1)

VEGFR-2/KDR (kinase domain receptor) also binds VEGF with high affinity.58 The key role of this receptor in developmental angiogenesis and hematopoiesis is evidenced by lack of vasculogenesis and failure to develop blood islands and organized blood vessels in VEGFR-2 null mice, resulting in death in utero between day 8.5 and 9.5.59 There is now agreement that VEGFR-2 is the major mediator of the mitogenic, angiogenic and permeability-enhancing effects of VEGF. VEGFR-2 undergoes dimerization and ligand-dependent tyrosine phosphorylation in intact cells and results in a mitogenic, chemotactic and and pro-survival signal. Several tyrosine residues have been shown to be phosphorylated (for review see ref.13). VEGF has been shown to induce the phosphorylation of several proteins in endothelial cells,60 including phospholipase C γ(PLCγ), PI3-K, ras GTPase activating protein (GAP),60 src family.61 VEGF induces endothelial cell growth by activating the Raf-MEK-ERK pathway.62 VEGF mutants which bind selectively to VEGFR-2 are fully active endothelial cell mitogens and permeabilityenhancing agents, whereas mutants which specifically bind VEGFR-1 are devoid of both activities.63 Furthermore, VEGFR-2 (but not VEGFR-1) activation has been shown to be required for the anti-apoptotic effects of VEGF for human umbilical vein endothelial cells (HUVEC).15 As previously noted, such a pro-survival effect of VEGF is mediated by the PI3-K/ Akt pathway.15

Neuropilin (NP)1 and NP-2 Earlier studies indicated that certain tumor and endothelial cells express cell surface VEGF binding sites distinct in affinity and molecular mass from the two known VEGF RTKs, VEGFR-1 and VEGFR-2.64 Interestingly, VEGF121 failed to bind these sites, indicating that exon-7 encoded basic sequences were required for binding to this putative receptor.64 Subsequently, Soker et al identified this isoform-specific VEGF receptor as NP-1,65 a molecule that had previously been shown to bind the collapsin/semaphorin family and was implicated in neuronal guidance (for review see ref. 66). When co-expressed in cells with VEGFR-2, NP-1 enhanced the binding of VEGF165 to VEGFR-2 and VEGF165-mediated chemotaxis.65 It has been proposed that NP-1 presents VEGF165 to the VEGFR-2 in a manner that enhances the effectiveness of VEGFR-2-mediated signal transduction.65 Binding to NP-1 may explain the greater mitogenic potency of VEGF165 relative to VEGF121. There is no evidence that NP-1 or the related NP-2 signals following VEGF binding.66 The role of NP-1 in the development of the vascular system has been demonstrated by gene targeting studies, documenting embryonic lethality in NP-1 null mice.67

VEGF and Its Receptors

5

Role of VEGF in Physiological Angiogenesis Embryonic and Early Postnatal Development

VEGF also plays an essential role in embryonic vasculogenesis and angiogenesis.68,69 Inactivation of a single VEGF allele in mice resulted in embryonic lethality between day 11 and 12. The VEGF+/- embryos exhibited a number of developmental anomalies, defective vascularization in several organs and a markedly reduced number of nucleated red blood cells within the blood islands in the yolk sac. VEGF also plays an important role in early postnatal life.18 Partial inhibition of VEGF achieved by inducible Cre-loxP- mediated gene targeting resulted in increased mortality, stunted body growth and impaired organ development. Administration of mFlt (1-3)-IgG, a fusion protein consisting of the first three Ig-like domains of mFlt-1 fused to a murine Fc, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest, when the treatment was initiated at day 1 or day 8 postnatally. Such treatment was also accompanied by rapid lethality, primarily due to kidney failure.18 Defective glomerular development in neonates was also observed in studies using anti-VEGF antibodies.70 The pivotal role of VEGF in kidney development is also demonstrated by very recent studies showing that selective VEGF deletion in podocytes leads to glomerular disease in a gene dosage-dependent fashion.71 Heterozygous mice developed renal disease by 2.5 weeks of age, characterized by proteinuria and endotheliosis. Homozygosity resulted in perinatal lethality.71

Skeletal Growth and Endochondral Bone Formation

Endochondral bone formation is a fundamental mechanism for longitudinal bone growth.72 VEGF mRNA is expressed by hypertrophic chondrocytes in the epiphyseal growth plate, suggesting that a VEGF gradient is needed for directional growth and cartilage invasion by metaphyseal blood vessels.73 Following VEGF blockade with a soluble VEGFR-1 chimeric protein or an anti-VEGF monoclonal antibody, blood vessel invasion is almost completely suppressed, concomitant with impaired trabecular bone formation, in mice and primates.73,74 Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption of hypertrophic chondrocytes was inhibited, resulting in a marked expansion of the hypertrophic chondrocyte zone. Importantly, cessation of the anti-VEGF treatment is followed by capillary invasion, restoration of bone growth and normalization of the growth plate architecture. A similar, although less dramatic phenotype was obtained, when VEGF was deleted in the cartilage of developing mice by means of Cre-loxP- mediated, tissue specific gene ablation.75

Ovarian Angiogenesis Follicular growth and the development of the corpus luteum (CL) are dependent on the proliferation of new capillary vessels. Subsequently, the blood vessels regress, suggesting the co-ordinated action of inducers and inhibitors of angiogenesis.76 Previous studies have shown that VEGF mRNA expression is temporally and spatially related to the proliferation of blood vessels in the ovary.77 Administration of VEGF inhibitors suppresses luteal angiogenesis74,78,79 and delays follicular development in rodents and primates.80 More recent studies have indicated that endocrine-gland derived VEGF (EG-VEGF), a novel selective angiogenic factor, plays a co-operative role with VEGF in the regulation of angiogenesis in the human ovary.81 EG-VEGF is not structurally related to VEGF but belongs to a unique gene family.82 A sequential activation of the two genes occurs in the CL.83 While VEGF is strongly expressed in early stage CL, its expression is reduced by mid-luteal phase. In contrast, EG-VEGF starts being expressed later than VEGF but persists throughout mid- and early-late luteal phase.83

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VEGF and Cancer

Role of VEGF in Pathologic Conditions Solid Tumors and Hematologic Malignancies In situ hybridization studies have demonstrated that VEGF mRNA is up-regulated in many human tumors (for review see refs. 7, 25). In 1993, Kim et al reported that anti-VEGF antibodies exert a potent inhibitory effect on the growth of several tumor cell lines in nude mice.84 Subsequently, many other tumor cell lines were found to be inhibited in vivo by this as well as other anti-VEGF treatments, including small molecule inhibitors of VEGFR signaling, antisense oligonucleotides and anti-VEGFR-2 antibodies (for review see ref. 7). While tumor cells represent the major source of VEGF, tumor-associated stroma is also an important site of VEGF production.85,86 Clinical trials in cancer patients are ongoing with several VEGF inhibitors, including a humanized anti-VEGF monoclonal antibody (rhu mAb VEGF),87 an anti-VEGFR-2 antibody,88 small molecules inhibiting VEGFR-2 signal transduction89 and a soluble receptor.90 Phase II clinical trial data have provided initial evidence that rhu mAb VEGF, in combination with conventional chemotherapy, results in increase in time to progression and even survival in patients with metastatic colorectal carcinoma.91 Thrombosis, hypertension, as well as some proteinuria, were among the side effects of such treatment. Furthermore, a randomized double blind placebo-controlled phase II trial has shown a highly significant increase in time to progression in renal cell carcinoma patients treated with rhu mAb VEGF as a single agent.92 In light of the fact that many renal cell carcinoma patients harbor mutations in the VHL gene, these results are particularly intriguing. rhu mAb VEGF treatment had modest toxicity in this study, primarily hypertension and asymptomatic proteinuria.92 Phase III studies are currently underway to confirm and fully assess the benefit of these anti-VEGF treatments in patients with advanced cancer. Most recently, Hurwitz et al have presented the result of a large randomized placebo-controlled phase III trial in metastatic colorectal cancer (manuscript submitted). Survival was significantly increased in patients in the chemotherapy (irinotecan, 5-fluorouracil, leucovorin) plus rhu mAb VEGF arm relative to chemotherapy alone. Interestingly, the increased incidence of thrombosis and proteinuria which was observed in phase II was not observed in this phase III study. Thus, the prolonged survival and improvement in other markers of clinical benefit observed with the addition of rhu mAb VEGF to standard chemotherapy confirms the importance of angiogenesis in the clinical outcome of patients with colorectal cancer.

Intra-Ocular Neovascular Syndromes Diabetes mellitus, occlusion of central retinal vein or prematurity with subsequent exposure to oxygen can all be associated with intra-ocular neovascularization, which may result in vitreous hemorrhages, retinal detachment, neovascular glucoma and blindness.93 All of these conditions are known to be associated with retinal ischemia. Elevations of VEGF levels in the aqueous and vitreous of eyes with proliferative retinopathy secondary to diabetes and other conditions have been previously described.94,95 Subsequent animal studies using various VEGF inhibitors have directly demonstrated the role of VEGF as a mediator of ischemia-induced intra-ocular neovascularization.96,97 Neovascularization and vascular leakage are also a major cause of visual loss in age-related macular degeneration (AMD), the overall leading cause of blindness.93 Earlier studies have demonstrated the immunohistochemical localization of VEGF in choroidal neovascular membranes from AMD patients.98 Currently, anti-VEGF strategies are being explored in clinical trials in AMD patients, using either a recombinant humanized anti-VEGF Fab antibody fragment (rhu Fab VEGF)99 or 2'-Fluoropyrimidine RNA oligonucleotide ligand (aptamers).100 rhuFab VEGF reduces angiogenesis and vascular leakage in a primate model of AMD.101 Both the aptamer and rhu Fab VEGF are currently in phase III trials.

VEGF and Its Receptors

7

Therapeutic Implications and Perspectives There is now little doubt that the VEGF family plays an essential role in the regulation of embryonic and postnatal physiologic angiogenesis processes, such as normal growth processes18,73 and cyclical ovarian function.78 Furthermore, VEGF inhibition has been shown to inhibit pathological angiogenesis in a wide variety of tumor models, leading to the clinical development of a variety of VEGF inhibitors. Clearly, a major question is what impact VEGF inhibition will have in human patients, especially those with highly advanced malignancies. This question will be answered by the several phase III clinical trials that are currently underway, targeting colorectal, lung and renal cell carcinomas. Initial results indicate that there is at least some reason for optimism. However, progression eventually occurs in many patients, raising the issue that pathways mediating angiogenic escape after VEGF inhibition exist. Different angiogenic mechanisms might be differentially important in different tumor types and at various stages of the neoplastic progression.4,82 The potential clinical utility of VEGF inhibition is not limited to cancer. Trials in AMD patients are already in phase III. Furthermore, as already noted, gynecologic conditions such as endometriosis or the polycystic ovary syndrome might benefit from this treatment. The ability of VEGF and other angiogenic factors to promote collateral vessel growth in various animals model of ischemia generated much enthusiasm and led to several clinical trials in patients with coronary or limb ischemia.5 So far, the clinical results with VEGF (or bFGF) have been somewhat disappointing because the treatment did not significantly increase exercise treadmill time, although some improvement in angina class was measured, in a placebo-controlled trial with recombinant VEGF165 in coronary ischemia patients.102 An increase in vascularity was reported in a controlled trial with adenovirus-mediated delivery of VEGF165 in limb ischemia patients.103 Currently, the possibility is being explored that a more persistent exposure than that achieved in an early trial, when only bolus and brief infusions were administered,102 may achieve better results. In this context, recent studies using a conditional VEGF switch have shown that early cessation of the VEGF stimulus results in regression of newly formed vessels. However, after a critical duration of exposure, the vessels persisted for months after VEGF withdrawal and resulted in improved organ perfusion.104 Also, a greater understanding of the differential role of the VEGF receptors may open additional avenues. In particular, recent studies have emphasized that VEGFR-1, a protein with complex roles, has important functions in hematopoiesis and in the recruitment of mononuclear cells. Furthermore, the recent report that a receptor VEGFR-1- selective mutant may protect the liver from toxic damage extends the potential clinical applications of VEGFR-1 agonists.57 Other activities of VEGF may have interesting clinical implications. For example, on the basis of the key role played by VEGF in bone angiogenesis and endochondral bone formation, the application of this factor might be useful to enhance revascularization in nonhealing fractures and other conditions. A recent study has shown that VEGF administration leads to enhanced blood vessel formation and ossification in models of bone damage.105 These findings raise hope that a return to human trials with a better molecular and biological understanding of blood vessel growth and differentiation may be more rewarding than the early attempts.

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84. Kim KJ, Li B, Winer J et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993; 362:841-844. 85. Fukumura D, Xavier R, Sugiura T et al. Tumor induction of VEGF promoter activity in stromal cells. Cell 1998; 94:715-725. 86. Gerber HP, Kowalski J, Sherman D et al. Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor. Cancer Res 2000; 60:6253-6258. 87. Presta LG, Chen H, O’Connor SJ et al. Humanization of an anti-VEGF monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997; 57:4593-4599. 88. Prewett M, Huber J, Li Y et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis. Cancer Res 1999; 59:5209-5218. 89. Wood JM, Bold G, Buchdunger E et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2000; 60:2178-2189. 90. Holash J, Davis S, Papadopoulos N et al. VEGF-Trap: A VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 2002; 99:11393-11398. 91. Kabbinavar F, Hurwitz HI, Fehrenbacher L et al. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003; 21:60-65. 92. Yang JC, Haworth L, Sherry RM et al. A randomized trial of bevacizumab, an anti-VEGF antibody, for metastatic renal cancer. N Engl J Med 2003; 349:427-434. 93. Garner A. Vascular diseases. In: Garner A, Klintworth GK, eds. Pathobiology of Ocular Disease. 2nd ed. NY: Marcel Dekker, 1994:1625-1710. 94. Aiello LP, Avery RL, Arrigg PG et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994; 331:1480-1487. 95. Malecaze F, Clemens S, Simorer-Pinotel V et al. Detection of vascular endothelial growth factor mRNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy. Arch Ophthalmol 1994; 112:1476-1482. 96. Aiello LP, Pierce EA, Foley ED et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 1995; 92:10457-10461. 97. Adamis AP, Shima DT, Tolentino MJ et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol 1996; 114:66-71. 98. Lopez PF, Sippy BD, Lambert HM et al. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci 1996; 37:855-868. 99. Chen Y, Wiesmann C, Fuh G et al. Selection and analysis of an optimized anti-VEGF antibody: Crystal structure of an affinity-matured Fab in complex with antigen. J Mol Biol 1999; 293:865-881. 100. Ruckman J, Green LS, Beeson J et al. 2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem 1998; 273:20556-20567. 101. Krzystolik MG, Afshari MA, Adamis AP et al. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol 2002; 120:338-346. 102. Henry TD, Annex BH, McKendall GR et al. The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation 2003; 107:1359-1365. 103. Makinen K, Manninen H, Hedman M et al. Vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: A randomized, placebo-controlled, double-blinded phase II study. Mol Ther 2002; 6:127-133. 104. Dor Y, Djonov V, Abramovitch R et al. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J 2002; 21:1939-1947. 105. Street J, Bao M, DeGuzman L et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA 2002; 99:9656-9661.

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CHAPTER 2

Hypoxic Regulation of VEGF Nina S. Levy, Ilana Goldberg-Cohen and Andrew P. Levy

Abstract

T

he induction of VEGF is an important step in the angiogenic response to hypoxia. Molecular studies have determined that VEGF is regulated primarily at the level of the mRNA. Specifically, hypoxia leads to an increase in the transcription of VEGF as well as an increase in the stability of the message. Two major proteins, which mediate these effects, are hypoxia inducible factor, HIF-1, and HuR, respectively. HIF-1 binds to the VEGF promoter and forms a complex that activates transcription of the VEGF gene. Hypoxia stimulates HIF-1 activity by inhibiting the rapid degradation of the HIF-1 α subunit via the ubiquitin proteosome pathway. The mechanism of HuR stabilization of VEGF mRNA is less well understood but appears to act by displacing RNases that mediate rapid degradation of VEGF mRNA. This review summarizes the work which led to these findings and highlights disease processes that may result from faulty hypoxic regulation of VEGF.

Introduction All tissues depend on an adequate supply of oxygen in order to maintain energy production and tissue function. Accordingly, adaptive mechanisms have evolved to cope with periods of decreased oxygen availability. A major mechanism of adaptation is the sprouting of new capillaries from the pre-existing network of blood vessels towards the hypoxic tissue. This process of angiogenesis occurs through stimulation of endothelial proliferation and maturation. VEGF is a potent endothelial-cell specific mitogen and a key mediator of angiogenesis. Accordingly, VEGF is rapidly induced in almost every cell type in response to hypoxia. Physiological situations in which VEGF is upregulated in response to low oxygen include wound healing, ovulation, and atherosclerosis. Inappropriate stimulation of VEGF can occur in pathological settings such as tumor growth, diabetic retinopathy, and diabetic nephropathy. Understanding the regulation of VEGF by hypoxia will contribute to the development of therapeutic strategies for modulating VEGF production depending on the physiological need.

Transcriptional Regulation of VEGF Identification of HIF-1 VEGF mRNA is increased by hypoxia in almost every cell system tested. Early mechanistic studies focused on increased transcription of the VEGF gene. The rate of VEGF transcription under hypoxic conditions was investigated in rat pheochromocytoma PC12 cells. Nuclear run-off studies showed an approximately three-fold increase in the rate of VEGF transcription.1 Cloning of rat VEGF sequences into a transient expression vector led to the delineation of a hypoxia responsive element in the 5' flanking region of the VEGF gene. Electromobility gel-shift assays (EMSA) confirmed that the hypoxia responsive element regulating VEGF is indistinguishable from the hypoxia-inducible factor 1 (HIF-1) binding site in sequence and protein binding characteristics. VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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HIF-1 was originally described in studies of the erythropoeitin (epo) gene, a glycoprotein hormone that binds to erythroid progenitor cells and enables them to differentiate into red blood cells, thereby increasing the oxygen carrying capacity of the blood.2 HIF-1 binds to a region in the 3' untranslated region (UTR) of the epo gene and acts as a transcriptional enhancer, upregulating epo levels 50-100 fold in response to hypoxia. It is now clear that activation of this transcription factor is a common pathway in the up-regulation of many hypoxia-regulated genes such as tyrosine hydroxylase, glucose transporter 1, and many glycolytic enzymes. The hypoxic regulation of VEGF in many respects has become the study of the hypoxic regulation of HIF-1.

Mechanisms of HIF-1 Regulation by Hypoxia Great progress has been achieved in the last few years in elucidating the mechanism of HIF-1 activation under hypoxic conditions.3 HIF-1 is a heterodimer composed of two subunits: HIF-1 α and HIF-1 β. Both subunits belong to the basic helix-loop-helix family of proteins, which contain a basic DNA binding domain, a heterodimerization domain, and regulatory domains. The hypoxic regulation of HIF-1 is mediated primarily by an oxygen-dependent degradation domain (ODD) unique to HIF-1 α. Under normoxic conditions, HIF-1 α is hydroxylated at two specific proline residues within this domain. The hydroxylated prolines trigger ubiquitin conjugation and proteosomal degradation of HIF-1 α. Low oxygen tension inhibits the prolyl hydroxylase, thereby stabilizing HIF-1 α and allowing it to interact with HIF-1 β. Full transcriptional activation of HIF–1 under hypoxic conditions requires not only its stabilization but also recruitment of and interaction with other constitutive transcription factors. HIF-1 α is known to bind to the general transcription cofactor p300/CBP (cAMP response element binding protein). Transfection studies using the gene for the adenovirus E1A protein, which inactivates p300/CBP, indicated that p300/CBP is necessary for the hypoxic induction of VEGF.4 In addition, reporter construct studies found that additional, potentiating sequences in the 5’UTR are necessary for full transcriptional activation of VEGF under hypoxia.1 The presence of a consensus sequence for activator protein – 1 (AP-1) in close proximity to the HIF-1 binding site led to EMSA studies which showed that oligonucleotides containing the AP-1 binding site were able to abolish binding of a hypoxia-inducible protein complex to the VEGF 5’UTR.5 AP-1 may thus interact with HIF-1 α to promote tighter binding of p300/ CBP. The binding of p300/CPB to HIF-1 is inhibited by hydroxylation of HIF-1 at an asparagine residue present near its carboxy terminus. Transcriptional activation by HIF-1 is therefore also sensitive to hypoxia and explains the role of p300/CBP in the hypoxic response. Additional binding sites for transcription factors SP-1 and AP-2 are present in the promoter for VEGF. These transcription factors act in the absence of hypoxia,6 however, due to the reducing environment present under conditions of low oxygen, a greater proportion of these factors are likely to be present in their active state7 which can enhance their ability to stimulate transcription of VEGF. Redox sensitive sites on HIF-1 α have also been reported. Support for this hypothesis comes from transfection studies with the redox protein thioredoxin (Trx-1),8 which showed that cells stably transfected with Trx-1 results in an increase in VEGF levels under hypoxia. Figure 1 summarizes the regulation of HIF-1 by hypoxia and its role in the transcriptional activation of VEGF.

Post Transcriptional Regulation of VEGF Identification of mRNA Stability Elements The steady state levels of VEGF mRNA in PC12 cells are stimulated approximately 12 fold by hypoxia, however, increased transcription accounts for only part of this response.1 The existence of a long 3' untranslated region (3’UTR) rich in AU sequences in the VEGF gene suggested the presence of mRNA elements known to affect stability. The half-life of VEGF mRNA measured by actinomycin D experiments is approximately 40 minutes under normoxic

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Figure 1. Schematic diagram of the regulation of VEGF by HIF-1 (adapted from Huang and Bunn3). Under normoxic conditions HIF-1 α is hydroxylated at specific proline (P) and asparagine (N) residues by prolyland asparagyl- hydroxylases (PH and NH respectively). N- hydroxylation inhibits interaction of HIF-1 α with the transcriptional co-activator CBP/300. P-hydroxylation targets binding of VHL, followed by assembly of a E3 ubiquitin ligase complex. Subsequent polyubiquitylation of HIF-1 α leads to proteosomal degradation. Under conditions of low oxygen HIF-1 α is stable and interacts with HIF-1 β to form a heterodimer. HIF-1 and AP-1 bind to specific sequences in the VEGF promoter and promote co-operative binding of CBP/300. Additional binding of transcription factors SP-1 and AP-2 in the VEGF promoter may augment hypoxic transcriptional activation.

conditions. Hypoxia resulted in a 2.5 fold increase in mRNA half-life, suggesting the induction of mRNA stabilizing factors by hypoxia.9 Recapitulation of the hypoxia-inducible increase in mRNA stability using reporter constructs has been difficult to achieve. Transfer of VEGF 3’UTR sequences to a luciferase construct conferred a decrease in activity under normoxia; however, hypoxia-inducible increases in activity were unobtainable even in the presence of the entire 3’UTR (unpublished observation). Interestingly, hypoxia-inducible mRNA stability was achieved for a VEGF construct only in the presence of the 5’UTR, coding, and 3’UTR sequences. These results indicate potential co-operation between the various mRNA elements, a phenomenon previously seen in the stabilization of other cytokine messages.10 In vitro mRNA degradation assays focusing on the 3’UTR of VEGF revealed the presence of stability as well as instability sequences.9 Deletion analysis of VEGF 3' UTR sequences identified two regions, which confer instability to VEGF mRNA. Both regions contain nonamer consensus sequences present in the 3' UTRs of other short-lived cytokine messages. In the presence of hypoxic nuclear extracts, a 500 bp region in the 3’UTR (nucleotides 1255-1754) that overlaps with one of the nonamers conferred an increase in VEGF mRNA half-life. These results led to the hypothesis that under hypoxic conditions, a hypoxia-inducible protein complex binds at instability sites and prevents coupling and subsequent action of the mRNA degradation machinery.

Hypoxic Regulation of VEGF

15

Characterization of HuR EMSA studies confirmed that a hypoxia-inducible complex binds to the stabilizing sequences identified in the RNA degradation assays.9 HuR, a member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, was investigated as a candidate member of the hypoxia-inducible stabilizing complex. Inhibition of HuR expression in human epithelial 293T cells using antisense RNA abrogated the hypoxia-inducible increase in VEGF mRNA stability.11 Likewise, over expression of HuR increased the stability of VEGF mRNA, but only in hypoxic cells, indicating the likely presence of other hypoxia-inducible factors in the stabilizing complex. In vitro mRNA degradation assays confirmed these results and showed that addition of HuR to VEGF mRNA confers an increase in mRNA half-life in the presence of hypoxic extracts. RNase T1 selection analysis and nitrocellulose filter binding assays showed that HuR binds with high affinity (Kd = 9 nM) and specificity to an AU-rich 45 bp element (nucleotides 1630-1675) within a VEGF regulatory segment that confers mRNA stability under hypoxia.12 A second study using rat skeletal muscle extracts in an RNA EMSA super shift assay confirmed binding of HuR to this region and suggested the existence of two HuR binding sites within this AU-rich element (nucleotides 1659-1665 and 1666-1671).13 A reporter assay was developed to monitor mRNA stability mediated by HuR. Segments of the VEGF 3’UTR were placed downstream of the luciferase gene and cotransfected with a second plasmid encoding HuR.12 Deletion analysis revealed a segment between nucleotides 1250 and 1400, which mediated increased reporter activity. Transfer of a 40 bp AU-rich element from this segment (nucleotides 1286-1325) to a luciferase reporter construct conferred a hypoxia-inducible increase in luciferase activity. RNA degradation assays confirmed that the increased luciferase activity was due to prolonged mRNA half-life. It is interesting to note that deletion of the HuR binding site(s) at nucleotides 1630-1675 (described in the preceding paragraph) did not result in a significant decrease in reporter activity. HuR may bind to this site but be unable to effect stabilization, or there may exist redundancy in HuR binding and stabilization. Lead protection and RNase T1 mapping revealed that HuR contacts a highly U-rich 16 bp sequence at the 3' end of the 40 bp element (nucleotides 1308-1323).12 Transfer of the minimal 16 bp HuR binding site to a reporter gene was not sufficient to confer hypoxic stabilization. In fact, removal of the 16 bp HuR binding site resulted in a message that is more unstable than the vector alone. Deletion of the HuR binding site appears to have converted the 40 bp RNA stabilizing element into a destabilizing element. Previous studies with HuR have identified an endonuclease that can cleave within an HuR binding site.14 Taken together, the results support a role for HuR in displacing enzyme complexes which rapidly degrade the mRNA. Figure 2 shows a schematic diagram of the hypoxic stabilization of VEGF mRNA by HuR. Several observations point to a novel mechanism in the regulation of HuR by hypoxia. Under normoxic conditions approximately 90% of HuR is located in the nucleus.15 Under conditions of cellular stress, such as heat shock or UV irradiation, cytoplasmic levels of HuR are increased several fold, however, the total cellular steady state level of HuR remains unchanged. An HuR nucleocytoplasmic shuttling sequence (HNS) has been described which allows HuR to travel between the two cellular compartments. Under hypoxia, HuR is thought to bind VEGF mRNA, thereby stabilizing the message and promoting its transfer to the cytoplasm.

Internal Ribosomal Entry Sites (IRES) The 5' untranslated region of VEGF is highly structured and unusually long (approximately 1.0 kb). In addition, one open reading frame encoding a tripeptide exists upstream of the AUG initiation codon for VEGF. According to the cap-dependent, ribosome-scanning model for translation, these features would be expected to inhibit efficient translation of VEGF. A cap-independent mechanism for translation initiation, originally described for a number of viral messages, has been described which involves internal sites of ribosome entry onto the

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VEGF and Cancer

Figure 2. Schematic diagram of the hypoxic stabilization of VEGF mRNA by HuR. The VEGF coding region is designated by an open box. Under normoxic conditions, AU-rich elements (hatched boxes) in the VEGF 3’UTR bind and recruit RNA endonucleases that lead to rapid degradation of the mRNA. Under hypoxic conditions, HuR binds to these destabilizing elements and forms a complex that protects the mRNA from rapid degradation. HuR may act by promoting export of the VEGF mRNA from the nucleus to the cytoplasm.

messenger RNA. These sites differ in primary sequence but appear share a similar secondary structure. Two independent IRESs have been described for the VEGF 5’UTR.16-18 Under hypoxic conditions, in which CAP-dependent translation becomes compromised, translation of VEGF may proceed unhindered. A recent gene therapy study showed that IRES sequences placed upstream of VEGF coding sequences reduced the amount of time needed to overcome ischemic injury.19 An IRES has also been described for the HIF-1 α gene.3

Abnormalities in the Hypoxic Regulation of VEGF von Hippel-Lindau (VHL) Protein Many tumor cells have been shown to overexpress VEGF, indicating the oncogenic potential of mutations affecting proteins regulating VEGF expression.20 One well-studied protein, whose mutated forms result in tumor growth is the von Hippel Lindau (VHL) protein. VHL is a tumor suppressor gene which, when inactivated, has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. Renal carcinoma cells lacking wild type VHL produce large amounts of VEGF mRNA in an oxygen independent manner.21 Reintroduction of the wild type VHL gene into these cells restores low normoxic levels of VEGF mRNA and reinstates the hypoxic induction of the message. Studies investigating the mechanism of VHL action indicated that the half-life of VEGF mRNA in cells containing mutated VHL was approximately 4 hrs under normoxic conditions. In cells containing wild type VHL the mRNA half-life was less than 1 hour.22 Likewise EMSA studies indicated that the hypoxia-inducible complex, which binds to the 3' UTR of VEGF

Hypoxic Regulation of VEGF

17

mRNA, is regulated in a parallel fashion to the expression of the gene. These studies suggest that VHL acts at the level of VEGF mRNA stabilization.21 More recent studies indicate that VHL plays a critical role in the transcriptional activation of VEGF. Specifically, VHL targets HIF-1 α for oxygen dependent proteolysis.3 VHL binds to at least one of two specific hydroxylated prolines on HIF-1 α, and together with elongin B, elongin C, and Cul2, functions as an E3 ubiquitin ligase for HIF-1 α polyubiquitylation. Cells lacking functional VHL show constitutively elevated levels of HIF-1 α. Consistent with these results is the finding that tumor mutations in VHL affect binding to HIF-1 α or to proteins in the ligase complex. These studies indicate that functional VHL protein is necessary for both increased transcription as well as increased mRNA stability of VEGF under hypoxic conditions. The mechanism of VHL mediated increases in VEGF mRNA stability is unclear; however, the fact that VEGF is not even partially hypoxia regulated in cells mutated for VHL lends support to its role in this pathway.

Inter-Individual Heterogeneity in the Hypoxic Induction of VEGF Collateral blood vessel growth in the heart is one example of the beneficial effects of hypoxia-induced VEGF. Interestingly, there is a wide range in the degree of collateral vessel development among patients with coronary artery disease (CAD). Only half of patients with CAD develop collaterals at all. One hypothesis is that failure to generate collateral vessels is associated with a failure to appropriately induce VEGF under ischemic conditions. This hypothesis was tested by correlating the VEGF response to hypoxia in monocytes harvested from CAD patients with the presence of collaterals visualized during routine angiography. The results showed that patients with no collaterals induced VEGF to a significantly lesser extent than patients with well developed collaterals.23 In a similar vein, it was found that diabetic patients who do not develop diabetic retinopathy also had a reduced induction of VEGF in response to hypoxia.24 Both of these studies indicate that failure to induce VEGF has a profound affect on the progression of vascular disease and suggests the involvement of other polymorphic loci involved in this process. Recently, a number of negative regulators of HIF-1 have been described. Examples include the cited2 protein which binds p300/CBP during embryonic development and competes with HIF-1 α, thereby interfering with hypoxia-driven transcription.25 A novel inhibitory PAS protein (IPAS), which is a splice variant of HIF-3 α, another member of the HIF α family of proteins, lacks the ability to activate transcription.26 IPAS is highly expressed in corneal epithelium where angiogenesis is suppressed despite tissue hypoxia. Another naturally occurring splice variant of HIF-1 α lacks exons 11 and 12 and results in a truncated protein lacking degradation and transcriptional activation domains.27 This variant competed with endogenous HIF-1 α and suppressed HIF-1 activity, resulting in the down-regulation of mRNA expression of hypoxia-inducible genes. HIF-1 α appears to be the master regulator of hypoxia driven gene expression in general and of VEGF in particular. It would be interesting to see if variants of HIF-1 can account for the individual heterogeneity seen in the hypoxic regulation of VEGF. Is this inter-individual heterogeneity in the hypoxic induction of VEGF relevant for tumor angiogenesis? One might predict that in those individuals in which VEGF is induced to a greater degree with hypoxia that a tumor would be more angiogenic and aggressive. Therefore, it is of considerable interest that there are differences in the transcriptional activation and stability of VEGF in numerous tumor cell lines.20,28

Acknowledgments This work was supported in part by the Kennedy Leigh Charitable trust and NIH RO1 HL66195 both to APL.

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References 1. Levy AP, Levy NS, Wegner S et al. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem 1995; 270:13333-13340. 2. Bunn HF, Gu J, Huang LE et al. Erythropoietin: A model system for studying oxygen-dependent gene regulation. J Exp Biol 1998; 201:1197-1201. 3. Huang LE, Bunn HF. Hypoxia-inducible factor and its biomedical relevance. J Biol Chem 2003; 278:19575-19578. 4. Arany Z, Huang LE, Eckner R et al. An essential role for p300/CBP in the cellular response to hypoxia. Proc Natl Acad Sci USA 1996; 93:12969-12973. 5. Damert A, Ikeda E, Risau W. Activator-protein-1 binding potentiates the hypoxia-induciblefactor1-mediated hypoxia-induced transcriptional activation of vascular-endothelial growth factor expression in C6 glioma cells. Biochem J 1997; 327:419-423. 6. Ryuto M, Ono M, Izumi H et al. Induction of vascular endothelial growth factor by tumor necrosis factor α in human glioma cells. Possible roles of SP-1. J Biol Chem 1996; 271:28220-28228. 7. Abate C, Patel L, Rauscher FJ et al. Redox regulation of fos and jun DNA-binding activity in vitro. Science 1990; 249:1157-1161. 8. Welsh SJ, Bellamy WT, Briehl MM et al. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1α protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis. Cancer Res 2002; 62:5089-5095. 9. Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996; 271:2746-2753. 10. Dibbens JA, Miller DL, Damert A et al. Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements. Mol Biol Cell 1999; 10:907-919. 11. Levy NS, Chung S, Furneaux H et al. Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol Chem 1998; 273:6417-6423. 12. Goldberg-Cohen I, Furneauxb H, Levy AP. A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR. J Biol Chem 2002; 277:13635-13640. 13. Tang K, Breen EC, Wagner PD. Hu protein R-mediated posttranscriptional regulation of VEGF expression in rat gastrocnemius muscle. Am J Physiol Heart Circ Physiol 2002; 283:H1497-H1504 14. Zhao Z, Chang FC, Furneaux HM. The identification of an endonuclease that cleaves within an HuR binding site in mRNA. Nucleic Acids Res 2000; 28:2695-2701. 15. Brennan CM, Steitz JA. HuR and mRNA stability. Cell Mol Life Sci 2001; 58:266-277. 16. Stein I, Itin A, Einat P et al. Translation of vascular endothelial growth factor mRNA by internal ribosome entry: Implications for translation under hypoxia. Mol Cell Biol 1998; 18:3112-3119. 17. Miller DL, Dibbens JA, Damert A et al. The vascular endothelial growth factor mRNA contains an internal ribosome entry site. FEBS Lett 1998; 434:417-420. 18. Huez I, Creancier L, Audigier S et al. Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA. Mol Cell Biol 1998; 18:6178-6190. 19. Roguin A, Avivi A, Nitecki S et al. Restoration of blood flow by using continuous perimuscular infiltration of plasmid DNA encoding subterranean mole rat Spalax ehrenbergi VEGF. Proc Natl Acad Sci USA 2003; 100:4644-4648. 20. White FC, Carroll SM, Kamps MP. VEGF mRNA is reversibly stabilized by hypoxia and persistently stabilized in VEGF-overexpressing human tumor cell lines. Growth Factors 1995; 12:289-301. 21. Levy AP, Levy NS, Goldberg MA. Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel-Lindau protein. J Biol Chem 1996; 271:25492-25497. 22. Iliopoulos O, Levy AP, Jiang C et al. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci USA 1996; 93:10595-10599. 23. Schultz A, Lavie L, Hochberg I et al. Interindividual heterogeneity in the hypoxic regulation of VEGF: Significance for the development of the coronary artery collateral circulation. Circulation 1999; 100:547-552. 24. Marsh S, Nakhoul FM, Skorecki K et al. Hypoxic induction of vascular endothelial growth factor is markedly decreased in diabetic individuals who do not develop retinopathy. Diabetes Care 2000; 23:1375-1380. 25. Yin Z, Haynie J, Yang X et al. The essential role of Cited2, a negative regulator for HIF-1α, in heart development and neurulation. Proc Natl Acad Sci USA 2002; 99:10488-10493. 26. Makino Y, Cao R, Svensson K et al. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 2001; 414:550-554. 27. Chun YS, Choi E, Kim TY et al. A dominant-negative isoform lacking exons 11 and 12 of the human hypoxia-inducible factor-1α gene. Biochem J 2002; 362:71-79. 28. Scott PA, Gleadle JM, Bicknell R et al. Role of the hypoxia sensing system, acidity and reproductive hormones in the variability of vascular endothelial growth factor induction in human breast carcinoma cell lines. Int J Cancer 1998; 75:706-712.

CHAPTER 3

Molecular Mechanisms of VEGF-Induced Angiogenesis Sandra Donnini, Marina Ziche and Lucia Morbidelli

Abstract

A

ngiogenesis is a complex process that occurs in a series of inter-related steps, and involves the release of pro-angiogenic factors. One of the most important angiogenic factors is vascular endothelial growth factor (VEGF). VEGF regulates both vascular endothelial cell migration, proliferation and permeability, and functions as an anti-apoptotic factor for newly formed blood vessels. The biological effects of VEGF are mediated by two receptors, VEGFR-1 and VEGFR-2, whose expression is mostly limited to the vascular endothelium. Angiogenesis, the role of VEGF in angiogenesis and the signal cascade regulating VEGF-induced angiogenesis are discussed.

Angiogenesis Angiogenesis, the sprouting of new vessels from existing vasculature, is a fundamental requirement for organ development and differentiation during embryogenesis, wound healing and the female reproductive cycle.1,2 Pathologic angiogenesis is characterized by either inadequate (e.g., coronary artery disease) or excessive neovascularization (e.g., rheumatoid arthritis, age-related macular degeneration, proliferative retinopathies and psoriasis as well as tumor growth and metastasis).1-5 Angiogenesis occurs in a series of complex and inter-related steps (Fig. 1). First, the early events required for sprouting of new capillaries include nitric oxide (NO)-mediated vasodilation of pre-existing capillaries and the release of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), from injured cells or cancer cells with activation of endothelial cells to initiate the degradation of underlying basement membrane. Endothelial derived relaxing factor or NO plays a pivotal role in the action of angiogenic growth factors. A number of angiogenic growth factors, including VEGF, or vasoactive peptides upregulate the expression and activity of endothelial constitutive NO synthase (eNOS) and stimulate the release of endothelium-derived NO. Activated endothelial cells produce enzymes such as metalloproteinases (MMPs), invade the extracellular matrix and migrate and divide in response to growth factors. Integrins, such as αvβ3 and αvβ5, mediate the migration of the new endothelial cells through the dissolved basement membrane towards the source of growth factors as well as the differentiation, maturation and survival of new blood vessels.6-8 Finally, endothelial cells organize into new tubes structurally supported by pericytes and able to carry blood to the tissue that initially released the pro-angiogenic growth factors.6,7,9

The Role of Vascular Endothelial Growth Factor in Angiogenesis VEGF plays a central role in all the processes of angiogenesis described above. It regulates vascular endothelial cell proliferation, migration, invasion, and permeability, and also functions as an anti-apoptotic factor for endothelial cells in newly formed vessels.10,11 The imporVEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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Figure 1. Angiogenesis: cascade of events. The process of angiogenesis occurs as an orderly series of events including vasodilation of pre-existing capillaries, degradation of basement membrane surrounding the existing blood vessels, migration of endothelial cells from preexisting vessels through the dissolved basement membrane, endothelial cell proliferation, tubule formation and connection in order to form blood vessel loops that can circulate blood. Finally, newly formed vessel tubes are stabilized (vessel maturation) by muscle cells or pericytes that provide structural support. The events are tightly controlled by a variety of molecules such as NO, VEGF, FGF-2, MMPs, and integrins (αvβ3 and αvβ5).

tance of VEGF-induced signaling has been demonstrated by genetic and pharmacological inactivation of its receptors which leads to a complete lack of blood vessel development in the embryo, and dramatically impairs the growth of cancer cells in vivo.12-14 At the moment there are six known members of the VEGF family: VEGF/VEGF-A, placental growth factors, (PlGF-1 and PlGF-2), VEGF-B, VEGF-C, VEGF-D, and VEGF-E.15 These glycoproteins belong to a structural superfamily of growth factors that includes platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor beta 1 (TGFβ1).16 VEGF is expressed by tumor cells, macrophages, T cells, smooth muscle cells, kidney cells, keratinocytes, astrocytes and osteoblasts.17 VEGF has mitogenic activity in vascular endothelium of arteries, veins and lymphatics, but not to an appreciable extent in other cell types.18 Malignant transformation of cultured cells often results in an induction of VEGF expression.19 Recently, constitutive expression of VEGF and its receptors was observed in most primary and metastatic melanoma cell lines.20 The most important mechanism regulating VEGF gene expression is hypoxia. Hypoxia-induced transcription of VEGF mRNA and protein is mediated by hypoxia-inducible factor-1 (HIF-1) binding to an element in the VEGF gene promoter21-24 and enhanced stability of VEGF mRNA.25 Other factors shown to up-regulate VEGF mRNA expression include cytokines such as epidermal growth factors (EGFs), PDGF-BB, TGFβ1, keratinocyte growth factor (KGF), interleukin-1 beta (IL-1β) and oncogenes.18,26 In addition to its role in the paracrine stimulation of angiogenesis, VEGF may also have an autocrine stimulatory effect on tumor cells.27 The biological effects of VEGF are mediated by three receptor tyrosine kinases (RTKs), VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and VEGFR-3 (Flt-4).17 The expression of these receptors is largely restricted to the vascular endothelium18,28 and it is assumed that all the effects of VEGF on endothelial cells are mediated by these receptors. Most biological functions of VEGF are mediated via VEGFR-2, whereas the role of VEGFR-1 is controversial and not fully elucidated. VEGFR-3 is mostly expressed on lymphatic endothelial cells and is involved in the stimulation and maintenance of lymphatic vessels.29 VEGFR-2 is almost exclusively expressed in endothelial cells and appears to mediate the dominant signal transduction

Molecular Mechanisms of VEGF-Induced Angiogenesis

21

Figure 2. The VEGF pathway. The figure represents a cross-sectional view of an endothelial cell and depicts the VEGF signalling pathway in angiogenesis. Binding of VEGF to its receptor, VEGFR-1 or VEGFR-2, results in recruitment of adapter molecules containing no intrinsic catalytic activity such as Shc, Grb2 and Nck which couple activated receptors to a cascade of intracellular pathways, leading to proliferation, migration and differentiation of endothelial cells. FAK, focal adhesion kinase, hsp90, heat shock protein 90, PI3-K, phosphatidylinositol 3'-kinase, ERK1/2, extracellular regulated kinase 1/2, JUNK, c-Jun N-terminal mitogen protein kinase, PLA, phospholipase A, PLC, phospholipase C, PKC, protein kinase C, eNOS, endothelial constitutive nitric oxide synthase.

pathway regulating angiogenesis and tumorigenesis.10 Many molecular studies have provided evidence for the role of VEGFR-2 in tumor vascularization. Millauer et al used a retrovirus encoding a dominant-negative mutant of the VEGFR-2 to prevent the growth of a transplanted glioblastoma tumor, demonstrating the biological relevance of VEGFR-2/VEGF for tumor associated angiogenesis in vivo and validating the targeting of the VEGFR-2 signaling pathway for the development of anti-angiogenic agents.30

The VEGF Signal Cascade in Angiogenesis When VEGF binds to its receptors, the pro-angiogenic signal is transmitted by these receptor tyrosine kinases (RTKs) to downstream proteins, initiating a complex signal cascade (Fig. 2). VEGF-induced stimulation of VEGFR-2 results in conformational changes in VEGFR-2, followed by dimerization and autophosphorylation on tyrosine residues.31-33 These tyrosine residues are targets for SH2, SH3, and other proteins or kinases containing phosphotyrosinebinding domains such as Shc, Grb2, c-Src, Nck, and two tyrosine phosphatases SHP-1 and SHP-2, which can dephosphorylate RTKs.34 The association between stimulated VEGFR-2 and these proteins controls the activation of different and inter-related signal transduction pathways which may be divided into: (a) VEGF-induced endothelial cell proliferation and survival involving the activation of mitogen-activated protein kinases, (MAPKs), protein kinase C (PKC), and the Akt pathway and (b) VEGF-induced endothelial cell migration, in which the induction of MMPs, the activation of focal adhesion kinase (FAK) and phosphatidylinositol 3'-kinase (PI3-K) are implicated. NO, as a mediator of endothelial cell

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functions in vivo, links the VEGF-receptor signaling network to all its biological effects. NO mediates vasodilation, proliferation, survival and migration of endothelial cells in response to VEGF (see following sections). VEGF stimulates DNA synthesis and proliferation in a variety of endothelial cell types by activation of VEGFR-2.35-38 Stimulated VEGFR-2 induces the activation of the serine-threonine mitogen activated protein kinase ERK1/2 (extracellular regulated kinase) which in turn, increases the activity of the c-Jun N-terminal kinase (JUNK) 38 both playing a central role in endothelial cell proliferation.35-37 ERK1/2 is required for JUNK activation and this occurs through ERK activating JUNK kinases, including SEK-1. In fact, expression of a dominant-negative SEK-1 mutant blocks either JUNK and ERK activity and cell growth.38 It has been reported that activation of ERK1/2 kinase is mediated by the Ras-Raf-MEK-ERK pathway36,38 or phospholipase A (PLA)-γ-induced PKC δ-activation of Raf-MEK-ERK pathway.39-41 Moreover, an alternative mechanism for VEGF-induced activation of Raf-MEK-ERK pathway in endothelial cells involving NO-mediated Raf-1 activation has been demonstrated.37 Two reports also show that VEGF promotes endothelial nitric oxide synthase (eNOS) activation involving two different isoforms of PKC, PKC-ε and –δ.42 VEGF induces angiogenesis not only by promoting endothelial cell proliferation, but also by inhibiting apoptosis by activation of Akt via the PI3-K-dependent pathway.43,44 VEGF has been shown to regulate the expression of several anti-apoptotic proteins, such as Bcl-2 and A1.44 These proteins in turn, inhibit the activation of caspases and up-regulate the expression of survivin and X-chromosome-linked IAP (XIAP), two inhibitors of apoptosis reported to inhibit small pro-domain caspases, the terminal pro-apoptotic effectors.45 Moreover, in vivo in a human glioma xenograft model it has been demonstrated that downregulating VEGF transgene expression results in the selective obliteration of immature blood vessels that have not recruited periendothelial cells.46 Similar results were observed when the production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy in human prostate cancer. These results show the pivotal role of VEGF in the growth and survival of newly formed vessels in tumors. VEGF induces endothelial cell migration by promoting different pathways, such as FAK and paxillin phosphorylation and PI3-Akt-eNOS.47,48 FAK activation is mediated by the C-terminal region of VEGFR-2 and PI3-K activation.47 Other pathways for VEGF-induced migration have been reported involving heat shock protein, Hsp90-mediated FAK phosphorylation48 or p38 MAPK activation.49 Using a specific p38 kinase inhibitor, VEGF-induced endothelial cell migration can be inhibited, whereas an ERK1/2 MAPK inhibitor does not affect this cellular function.49 Ziche et al and others have demonstrated that NO plays a role in VEGF-induced migration.50-52 VEGF promotes NO production by Akt-mediated phosphorylation of eNOS in endothelial cells and NO is implicated in endothelial cell migration.48 Moreover, it has also been reported that NO regulates focal adhesion formation and phosphorylation of FAK in endothelial cells.52 NO production by VEGF is mediated by VEGFR-2 activation and it is implicated in mediating different biological effects of the growth factor including angiogenesis and vascular permeability.53,54 VEGFR-2 also mediates upregulation of eNOS and iNOS (inducible nitric oxide synthase) mRNAs via a PKC pathway.55 VEGF induces Akt activation which in turn mediates phosphorylation of eNOS in a Ca2+-independent manner.48,56 This pathway together with the up-regulation of eNOS mRNA via PKC represent important mechanisms controlling long-term NO production by VEGF which may contribute to the biological functions of Akt signalling such as survival and migration. Contrasting data have been reported for the VEGFR-1 selective agonist, PlGF. Some reports demonstrated that it does not stimulate either proliferation or migration in endothelial cells.32,57 Conversely, in 1997, Ziche et al demonstrated that PlGF-1 promotes angiogenesis in vivo and induces endothelial cell migration and proliferation in vitro.58 The best characterized responses mediated by VEGFR-1 are the stimulation of monocyte migration and tissue factor

Molecular Mechanisms of VEGF-Induced Angiogenesis

23

expression in monocytes and endothelial cells.59,60 Recently, VEGFR-1 expression has been demonstrated on smooth muscle cells,61 where it controls MMP expression and production, and, in hypoxic conditions, smooth muscle cell proliferation and vasoconstriction.62 More direct evidence for the role of VEGFR-1 has come from studies using a soluble form of an extracellular domain of VEGFR-1 (sFlt-1). This peptide forms inactive complexes with VEGF or VEGFR-2 and inhibits VEGF-induced endothelial cell migration and proliferation.63,64 Moreover, exchanging the juxtamembrane region of the VEGFR-2 with VEGFR-1 suppresses VEGFR-2-mediated signaling and migration.65

Concluding Remarks Despite the recent progress in understanding the intracellular mechanisms mediating VEGFs biological functions in endothelium, there are several points in the VEGF signal transduction pathways that remain to be elucidated. Among these are: the identification of the molecules involved in the signaling by which VEGF mediates the production of NO and the characterization of their role in angiogenesis and tumorigenesis; the identification of the molecules relaying VEGFR-2 intracellular signal to the nucleus in order to identify the mechanisms by which endothelial cells control their phenotype during development, angiogenesis and tumorigenesis; and the role of VEGFR-1 in angiogenesis.

Acknowledgements This work was supported by funds from Italian Ministry for University (MIUR), the Italian Association for Cancer Research (AIRC) and the University of Siena (PAR 2002).

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18. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997; 18:4-25. 19. Kieser A, Weich HA, Brandner G et al. Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene 1994; 9:963-969. 20. Graeven U, Fiedler W, Karpinski S et al. Melanoma-associated expression of vascular endothelial growth factor and its receptors FLT-1 and KDR. J Cancer Res Clin Oncol 1999; 125:621-629. 21. Shweiki D, Itin A, Soffer D et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359:843–845. 22. Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996; 271:2746–2753. 23. Tuder R, Flook BE, Voelkel NF. Increased gene expression for VEGF and the VEGF receptors KDR/Flk and flt in lungs exposed to acute and chronic hypoxia. Modulation of gene expression by nitric oxide. J Clin Invest 1995; 95:1798–1807. 24. Carmeliet P, Dor Y, Herbert J et al. Role of HIF-1 alpha in hypoxia mediated apoptosis, cell proliferation, and tumor angiogenesis. Nature 1998; 394:485–490. 25. Ikeda E, Achen MG, Breier G et al. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J Biol Chem 1995; 270:19761-19766. 26. Neufeld G, Cohen T, Gengrinovitch S et al. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13:9-22. 27. Liu B, Earl HM, Baban D et al. Melanoma cell lines express VEGF receptor KDR and respond to exogenously added VEGF. Biochem Biophys Res Commun 1995; 217:721-727. 28. Kanno S, Oda N, Abe M et al. Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells. Oncogene 2000; 19:2138-2146. 29. Kubo H, Cao R, Brakenhielm E et al. Blockade of vascular endothelial growth factor receptor-3 signaling inhibits fibroblast growth factor-2-induced lymphangiogenesis in mouse cornea. Proc Natl Acad Sci USA 2002; 99:8868-73. 30. Millauer B, Shawver LK, Plate KH et al. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 1994; 367:576-579. 31. Meyer M, Clauss M, Lepple-Wienhues A et al. A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases. EMBO J 1999; 18:363–374. 32. Waltenberger J, Claesson-Welsh L, Siegbahn A et al. Differential signal transduction properties of KDR and Flt-1, two receptors for vascular endothelial growth factor. J Biol Chem 1994; 269:26988– 26995. 33. Heldin CH. Dimerization of cell surface receptors in signal transduction. Cell 1995; 80:213–223. 34. Kroll J, Waltenberger J. Regulation der Endothelfunktion und der Angiogenese durch den Vaskularen EndothelialenWachstumsfaktor-A (VEGF-A). Z Kardiol 2000; 89:206–218. 35. Abedi H, Zachary I. Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells J Biol Chem 1997; 272:15442–15451. 36. Kroll J, Waltenberger J. The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J Biol Chem 1997; 272:32521– 32527. 37. Parenti A, Morbidelli L, Cui X-L et al. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular-signal regulated kinase1/2 activation in postcapillary endothelium. J Biol Chem 1998; 273:4220–4226. 38. Pedram A, Razandi M, Levin E. ERK/JUNkinase cross-talk underlies vascular endothelial cell growth factor-induced endothelial cell proliferation. J Biol Chem 1998;273:26722–26728. 39. Doanes AM, Hegland DD, Sethi R et al. VEGF stimulates MAPK through a pathway that is unique for receptor tyrosine kinases. Biochem Biophys Res Commun 1999; 255:545–548. 40. Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells. Oncogene 1999; 18:2221–2230. 41. Gliki G, Abu-Ghazaleh R, Jezequel S et al. Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC-δ and by moblization of intracellular Ca2. Biochem J 2001; 353:503–512. 42. Shizukuda Y, Tang S, Yokota R et al. Vascular endothelial growth factor-induced endothelial cell migration and proliferation depend on a nitric oxide-mediated decrease in protein kinase C delta activity. Circ Res 1999; 85:247-56.

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43. Gerber HP, McMurtrey A, Kowalski J et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3-kinases/Akt signal transduction pathway. Requirement of Flk-1/KDR activation. J Biol Chem 1998; 273:30336–30434. 44. Gerber HP, McMurtrey A, Kowalski J et al. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998; 273:13313–13316. 45. Tran J, Rak J, Sheehan C et al. Marked induction of the IAP antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. Biochem Biophys Res Commun 1999; 264:781–788. 46. Benjamin LE, Keshet E. Conditional switching of vascular growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemanglioblastoma-like vessels by VEGF withdrawal. Proc Natl Acad Sci USA 1997; 94:8761-8766. 47. Qi, JH, Claesson-Welsh L. VEGF-induced activation of phosphoinositide 3-kinase is dependent on focal adhesion kinase. Exp Cell Res 2001; 263:173–182. 48. Dimmeler S, Fleming I, Fisslthaler B et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 1999; 399:601–605. 49. Rousseau S, Houle F, Kotanides H et al. Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase. J Biol Chem 2000; 275:10661–10672. 50. Ziche M, Morbidelli L, Masini E et al. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest 1994; 94:2036-44. 51. Noiri E, Hu Y, Bahou WF et al. Permissive role of nitric oxide in endothelin-induced migration of endothelial cells. J Biol Chem 1997; 272:1747–1752. 52. Goligorsky MS, Abedi H, Noiri E et al. Nitric oxide modulation of focal adhesions in endothelial cells. Am J Physiol 1999; 276:C1271–C1281. 53. Kroll J, Waltenberger J. VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2. Biochem Biophys Res Commun 1998; 252:743–746. 54. Zachary I, Mathur A, Yla-Herthuala S et al. Vascular protection: A novel, nonangiogenic cardiovascular role for vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 2000; 20:1512– 1520. 55. Shen B-Q, Lee DY, Zioncheck TF. Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and protein kinase C signaling pathway. J Biol Chem 1999; 274:33057–33063. 56. Fulton D, Gratton J-P, McCabe TJ et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 1999; 399:597–601. 57. Cunningham SA, Tran TM, Arrate MP et al. KDR activation is crucial for VEGF(165)-mediated Ca2+ mobilization in human umbilical vein endothelial cells. Am J Physiol 1999; 276:C176-181. 58. Ziche M, Maglione D, Ribatti D et al. Placenta growth factor-1 (PlGF-1) is chemotactic, mitogenic and angiogenic. Lab Invest 1997; 76:517-531. 59. Barleon B, Sozzani S, Zhou D et al. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996; 87:336–3343. 60. Clauss M, Weich H, Breier G et al. The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J Biol Chem 1996; 271:17629–17634. 61. Wang H, Keiser JA. Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: Role of flt-1. Circ Res 1998; 83:832-40 62. Parenti A, Brogelli L, Filippi S et al. Effect of hypoxia and endothelial loss on vascular smooth muscle cell responsiveness to VEGF-A: Role of Flt-1/VEGF-receptor-1. Cardiovasc Res 2002; 55:201-212. 63. Kendall R, Thomas K. Inhibition of vascular endothelial growth factor activity by an endogenousl y encoded soluble receptor. Proc Natl Acad Sci USA 1993; 90:10705–10709. 64. Roeckl W, Hecht D, Sztajer H et al. Differential binding characteristics and cellular inhibition by soluble VEGF receptors 1 and 2. Exp Cell Res 1998; 241:161–170. 65. Rahimi N, Dayanir V, Lashkari K. Receptor chimeras indicate that the vascular endothelial growth factor receptor 1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. J Biol Chem 2000; 275:16986–16992.

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CHAPTER 4

Crosstalk between VEGF and Bcl-2 in Tumor Progression and Angiogenesis Donatella Del Bufalo, Daniela Trisciuoglio and Michele Milella

Abstract

T

he study of genes involved in angiogenesis allows clarification of molecular and cellular events related to this phenomenon, as well as identification of new prognostic markers and new targets for cancer therapy. In this review we describe the relevance of the bcl-2 gene in the expression of vascular endothelial growth factor (VEGF) in endothelial and tumor cells and the importance of bcl-2 in relation to angiogenic potential. In particular, the angiogenic potential of tumor cells overexpressing bcl-2, the role of bcl-2 in VEGF-promoted survival of tumor cells, the role of bcl-2 in endothelial cells, and the clinical relevance of VEGF and bcl-2 expression will be discussed.

Introduction It is now established that angiogenesis plays an important role in the growth of solid and hematological tumors.1-3 Endothelial cells may support tumor cell growth through formation of new blood vessels and the release of different cytokines, and tumor cells in turn support endothelial growth by releasing VEGF or other pro-angiogenic factors.4 The main role of VEGF in tumor growth has been attributed to the induction of angiogenesis.5 VEGF regulates differentiation, migration and proliferation of endothelial cells by interacting with its receptors.6,7 VEGF has also been recently shown to be a survival factor for both endothelial8-13 and tumor cells,14-16 preventing apoptosis through the induction of bcl-2 expression. Moreover, bcl-2 has been shown to induce VEGF expression in different tumor histotypes.17-19 In addition to follicular B-cell lymphoma, where bcl-2 was firstly discovered,20 bcl-2 levels are elevated in a broad range of other cancers, including carcinomas of the breast, prostate, ovary, colon, lung, and melanoma, where it may have distinct biologic roles in cell survival, tumor progression, and drug resistance.21-23

Angiogenic Potential of Tumor Cells Overexpressing Bcl-2 Some years ago we demonstrated that bcl-2 overexpression enhances the metastatic potential of the human breast cancer line MCF7 ADR by inducing an increase in cell invasion, migration, and gelatinase production in vitro.24 Other groups have also demonstrated the involvement of bcl-2 in tumorigenicity, invasion, migration, and metastasis of different tumor histotypes.25-31 The fact that tumors derived from cells that overexpress bcl-2 grow more aggressively in vivo has been attributed by several authors to the anti-apoptotic properties of bcl-2.26,28 Since growth, progression, and metastasis of cancer are angiogenesis-dependent processes,1-3 we and other groups have evaluated whether the overexpression of bcl-2 can be associated with the development of or increase in the angiogenic phenotype.17-19 Several papers from our group and others have demonstrated a role for bcl-2 in angiogenesis in three different tumor VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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histotypes.17-19 Using bcl-2 overexpressing clones, obtained after transfection of breast MCF7 ADR, melanoma M14 and prostate carcinoma PC3, LNCaP, and DU-145 cell lines, it was found that bcl-2 overexpression substantially increases VEGF expression.17-19 An enhancement of VEGF protein secretion in vitro was observed in the breast, melanoma and two prostate carcinoma lines overexpressing bcl-2 when grown under hypoxic conditions. It is interesting to note that, in normoxic conditions, the breast carcinoma, the melanoma and the three prostate cell lines responded differently to bcl-2 overexpression. In particular, bcl-2 overexpression caused a strong increase in VEGF secretion in the DU-145 cells, a moderate increase in VEGF secretion in PC3 cells, and had no effect on VEGF secretion in LNCaP, M14 and MCF7 ADR cells. As suggested by the study on prostate carcinoma,18 it is possible that bcl-2 interacts with another factor(s) to regulate VEGF secretion, and that these factors are expressed constitutively in some cell lines (PC3, DU-145) but only in response to hypoxia in others (M14, MCF7 ADR, LNCaP). In all tumor histotypes analysed, the involvement of other pro-angiogenic factors in the bcl-2-mediated increase in angiogenesis was ruled out; in fact, the levels of hypoxia-induced basic fibroblast growth factor (bFGF) in breast, melanoma and prostate carcinoma, or transforming growth factor β-1 (TGFβ-1) in breast carcinoma were similar in control and bcl-2 transfectants. This observation suggests that bcl-2 overexpression specifically affects VEGF expression in response to hypoxia. Bcl-2 modulation of VEGF expression did not appear to be due to the ability of bcl-2 to protect breast, melanoma and prostate carcinoma cells from hypoxia-induced apoptosis, as hypoxia was not found to induce apoptosis in MCF7 ADR cells and no differences in apoptosis were observed after injection in nude mice of the breast carcinoma lines with different bcl-2 protein levels. PC3 and M14 were inherently resistant to hypoxia-induced apoptosis, regardless of their bcl-2 expression status. However, bcl-2 functionality was demonstrated by its ability to inhibit apoptosis triggered by stimuli other than hypoxia in all cell types studied.18,19,32 Thus, bcl-2 expression in these three cell lines does not have an indirect effect on hypoxia-induced secretion of VEGF by increasing resistance to hypoxia-induced apoptosis so that the lifespan of the tumor cells is lengthened and the secretion of the angiogenic factor is prolonged. Under hypoxic conditions, bcl-2 has a direct effect on VEGF expression that is independent of its anti-apoptotic activity. Such a clear distinction cannot be made in LNCaP cells, in which overexpression of bcl-2 affords increased resistance to hypoxia-induced apoptosis. Therefore, the increase in hypoxia-induced VEGF secretion in bcl-2 overexpressing LNCaP cells could either be due to a direct bcl-2 effect on VEGF or a consequence of bcl-2 anti-apoptotic activity. The observation that DU-145 cells overexpressing bcl-2 secrete more VEGF than control cells when cultured in normoxic conditions suggests that bcl-2 may directly regulate VEGF expression in this cell system. An increase in angiogenic potential of tumor cells after bcl-2 transfection was also observed using different in vivo assays such as matrigel, chicken chorioallantoic membranes (CAM) and mouse cornea.17-19 In particular, an enhanced neovascularization was found when matrigel or CAM containing supernatants from bcl-2 overexpressing breast and melanoma cells were compared to those containing supernatants from parental lines.17,19 Measuring the ability of prostate tumors derived from parental or bcl-2 overexpressing PC3 cells to induce neovascularization after implantation into corneal micropockets also confirmed the stronger angiogenic potential of bcl-2 transfectants.18 Increased VEGF expression and neovascularization were also observed in the bcl-2-overexpressing breast and prostate xenografts when compared to the tumors derived from parental cells.17,18 The use of VEGF specific antibodies validated the role of VEGF in bcl-2-induced in vitro and in vivo angiogenesis.19 Since several tumor cells express functional VEGF receptors,33-35 the generation of an autocrine loop that promotes tumor proliferation and migration can be hypothesized as a consequence of bcl-2 overexpression. Studies on bcl-2 overexpressing prostate tumors also demonstrated that apoptosis-resistant tumor cells give rise to tumors that are resistant to treatment with apoptosis-inducing agents, such as mitomycin C, while still being responsive to angiogenesis inhibitors, such as TNP-470, which targets the endothelial cells in the tumor vasculature.18

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Studies were also performed to provide mechanistic details into how bcl-2 influences VEGF expression.18,19 In the prostate carcinoma study it has been suggested that a mechanism other than the regulation of the levels of hypoxia inducible factor-1α (HIF-1α), a transcription factor involved in hypoxia-induced VEGF expression,36 is responsible for the stimulatory effects of bcl-2 on VEGF secretion in PC3 cells under hypoxic conditions. In fact, HIF-1α protein was expressed at similar levels in the bcl-2 overexpressing and control PC3 cells cultured in normoxic conditions and exposure to hypoxic conditions resulted in a similar induction of HIF-1α in both cell types.18 We have recently studied the two major control points for hypoxic induction of the VEGF: transcriptional regulation of its expression at the 5'-promoter and regulation of the steady-state level of mRNA.37,38 We demonstrated that HIF-1-mediated transcriptional activity and VEGF mRNA stabilization are two important control points in bcl-2/hypoxia induced VEGF expression in M14 melanoma cells.19 In particular, the half-life of VEGF message was longer in bcl-2 transfectants than in M14 control cells. In addition, bcl-2 overexpression increased VEGF promoter activity through the HIF-1 transcription factor. Enhanced HIF-1α protein expression and DNA binding activity were detected in bcl-2 overexpressing cells compared to M14 control cells. The discrepancy between our results and those of Fernandez et al on the role of HIF-1α in bcl-2-induced VEGF expression, may be due to the different approach used to evaluate HIF-1α. For several transcription factors no correlation has been demonstrated between expression at the protein level and DNA binding activity.39 Therefore, electrophoretic mobility shift assay may be a better method to evaluate the activity of transcription factors. The different histotypes, namely prostate PC3 and melanoma M14, in which the analysis has been performed should also be taken into account. Indeed, different cells can activate different pathways in order to upregulate VEGF in response to bcl-2 overexpression. Overall, these results are consistent with a model in which bcl-2 overexpression enhances the metastatic potential of different tumor histotypes by increasing tumor angiogenesis.26-31 In particular, increased metastasis after bcl-2 overexpression was observed in murine melanoma cells,26 and breast carcinoma cells24,31 and bcl-2 overexpression was associated with the development of androgen-independent prostate carcinomas.40 Several explanations exist for the role of bcl-2 protein in the acquisition of the angiogenic phenotype. We suggest that bcl-2 overexpression, which increases tumorigenicity and metastatic potential of several tumor lines,26-31 can stimulate a switch to the angiogenic phenotype in response to low oxygen conditions. Since hypoxia represents a characteristic of solid tumors, this microenvironmental stress may provide a common signal that induces a prolonged increase in angiogenic gene expression during tumorigenesis.41 Thus, if tumor hypoxia primes cells for increased VEGF expression, bcl-2 overexpression would provide the necessary signal to increase or maintain the state of angiogenic growth factor production. The possibility that bcl-2 could increase angiogenesis through downregulation of p5342 and consequent upregulation of VEGF,43 was excluded by studies on breast carcinoma, which showed no modulation of p53 protein expression after bcl-2 transfection.17 It is also possible that the VEGF increase induced by bcl-2 under hypoxic conditions, could enhance expression and/or activity of proteinases involved in angiogenesis.44-46 Regarding the possible mechanisms by which bcl-2 induces HIF-1α-mediated VEGF transcription and VEGF mRNA stabilization we formulated several hypotheses. Since we previously demonstrated that bcl-2 overexpression inhibits mitochondrial metabolism32 and HIF-1α hydroxylase (which regulates HIF-1α expression) is inactive in hypoxia,47 it is possible that bcl-2 protein can directly or indirectly modify post-translational hydroxylation of HIF-1α. It is also possible that bcl-2 acts on the expression of sequence-specific RNA binding proteins responsible for VEGF mRNA stabilization.48-51 Bcl-2 could also increase VEGF expression by stimulating the activity of transcription factors other than HIF-1. Indeed, we have previously demonstrated that bcl-2 increases nuclear factor κB (NF-κB) transcriptional activity in the MCF7 ADR line.52 Since NF-κB signaling blockade has been demonstrated to inhibit in vitro and in vivo expression of VEGF,53 it is

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possible that bcl-2 affects VEGF expression through modulation of the activity of NF-κB or other transcription factors. Together these data indicate that regulation of VEGF secretion is a new function for the bcl-2 oncogene, with important implications concerning the ability of bcl-2 to contribute to tumor progression. The bcl-2 gene can support tumor progression by both inhibiting apoptosis and inducing angiogenesis and, as suggested by Fernandez et al, it can be considered a more effective oncogene than those affecting only one of the processes involved in tumor progression such as proliferation, angiogenesis or apoptosis.18 Thus, the inhibition of bcl-2 by antisense oligonucleotides or small molecules might have multiple effects on tumor, by inhibiting angiogenesis as well as restoring sensitivity to antineoplastic agents or inducing apoptosis.54-57 Indeed, a recently published phase I/II clinical study reported encouraging results with the use of bcl-2 antisense oligonucleotides (G3139) in combination with dacarbazine in patients with advanced melanoma and a phase III randomized trial is ongoing.58 Additional controlled trials with 3139 are ongoing in other tumor types and have the goal of increasing the effectiveness of conventional treatment strategies.59

Role of Bcl-2 in VEGF-Promoted Survival of Tumor Cells Bcl-2 is also involved in the ability of VEGF to function as a survival factor for tumor cells.14-16 Even though VEGF has typically been considered an endothelial cell specific survival factor, it may also act as a survival factor for tumor cells, through induction of bcl-2 expression and inhibition of tumor cell apoptosis.14-16 VEGF treatment indeed promoted survival of neuroblastoma, mammary adenocarcinoma and leukemic cell lines through upregulation of bcl-2 expression, and anti-VEGF antibodies reduced bcl-2 expression and increased apoptosis.14-16 These alterations in bcl-2 expression were reflected by the levels of tumor cell apoptosis induced by several stimuli, such as serum starvation, tumor necrosis factor alpha (TNF-α) or geldanamycin. VEGF resulted in reduced tumor cell apoptosis, whereas its inhibition with anti-VEGF neutralizing antibodies induced apoptosis directly in tumor cells. The molecular mechanism by which VEGF increases the survival of leukemic cells has been characterized in the study on leukemic cells which focused on bcl-2 regulation.15 In particular, the authors demonstrated that VEGF induces heat shock protein 90 (Hsp90) and bcl-2 expression in VEGF receptor positive primary leukemias and cell lines. Upon VEGF stimulation, Hsp90 binds bcl-2 and apoptotic protease-activating factor-1 (APAF-1), an effect mediated through VEGF receptor-2 (KDR) and involving the activation of the mitogen activated protein kinase (MAPK) pathway. Conversely, geldanamycin, an antibiotic that selectively inhibits the activities of Hsp90, blocks VEGF-induced APAF-1 binding to Hsp90. Whether there is a direct interaction between Hsp90 and bcl-2, leading to inhibition of apoptosis, is still not established. Because Hsp90 stabilizes different proteins, thereby preventing their degradation, the authors proposed a model in which, in response to exogenous or endogenous VEGF, Hsp90 would prevent the degradation of bcl-2 and APAF-1, resulting in increased survival of VEGF receptor-expressing cells.15 This is not the case for the study on mammary carcinoma cells, because these cells are negative for the expression of KDR/Flk-1 or Flt 1 receptors.14 Thus, the signalling pathway through which VEGF acts on tumor cells can be different depending on cell type and remains to be clarified. Altogether, these studies demonstrate that, in addition to its role in angiogenesis and vessel permeability, VEGF acts as a survival factor for tumor cells, inducing bcl-2 expression and inhibiting apoptosis. Thus, VEGF may promote tumor survival not only through angiogenesis, but also by altering apoptosis and its regulating proteins. A role for VEGF in preventing tumor cell apoptosis is further supported by reports demonstrating that overexpression of soluble neuropilin 1, which prevents VEGF binding to cell surface receptors, in tumor cells was associated with increased tumor cell apoptosis in vitro.60

Role of Bcl-2 in Endothelial Cells VEGF stimulates endothelial cell proliferation and chemotaxis and potently induces angiogenesis and vascular permeability.6,7 This angiogenic factor also functions as a survival factor

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for the endothelial cells in adult and embryonic blood vessels.8-13 VEGF is able to enhance the survival of human endothelial cells grown in a reduced nutrient environment in vitro or exposed to the apoptosis-inducing cytokine TNF-α or to the anti-angiogenic factor thrombospondin-1. 11,13 In vivo studies have shown that hormonal ablation leads to down-regulation of VEGF and destruction of immature microvessels in hormone-dependent tumors.10 This suggests that tumor endothelial cells require a constant input of survival signals from the tumor to remain viable, and that removal of these signals results in disruption of the tumor capillary bed. The angiogenic activity attributed to VEGF may be due in part to its ability to enhance endothelial cell survival by inducing expression of bcl-2.11,13 Enhanced survival of human dermal microvascular endothelial cells (HDMEC) exposed to VEGF or human umbilical endothelial cells (HUVEC) infected with an adenovirus expressing human VEGF165 is associated with a dose-dependent increase in bcl-2 expression11,13,15,60 and a decrease in the expression of the processed forms of the cysteine protease, caspase-3, or an increase in Hsp90 expression.13,15 This suggests that bcl-2 functions synergistically with VEGF to generate a potent and sustained angiogenic response, and that the ability of growth factors to promote and sustain angiogenesis may depend on whether they are also able to promote endothelial cell survival. The ability of bcl-2 to potentiate endothelial angiogenesis has also been demonstrated.13,61 Several papers have provided evidence supporting the hypothesis that apoptosis may be involved in angiogenesis, although some discrepancy exists: activation of angiogenesis is indeed related to inhibition of the apoptotic pathway in some reports,13,61 and to activation of apoptosis in others.62-67 It has been reported that HDMEC overexpressing bcl-2 proliferate at a rate comparable to parental cells exposed to VEGF and demonstrate the ability to align and differentiate into sprouts in vitro without the addition of VEGF. In addition, incorporation of these cells into poly L-lactic acid sponges and implantation into severe combined immunodeficient (SCID) mice, results in a significant enhancement of neovascularization and decrease in apoptotic cells as compared with control implants.13 In order to study how bcl-2 overexpression in endothelial cells modulates angiogenesis and affects tumor growth, the same authors used a SCID mouse model of human angiogenesis in which sponges seeded with oral squamous carcinoma or Kaposi’s sarcoma cells were implanted together with endothelial cells into SCID mice to generate human tumors vascularized with human microvessels.61 They demonstrated that overexpression of bcl-2 in tumor microvascular endothelial cells results in high intra-tumoral vascular density and accelerates tumor growth in vivo. Furthermore, the potentiation of angiogenic response associated with overexpression of endothelial bcl-2 is not only attributable to enhanced survival of these cells but is also mediated by the resulting synthesis of the endothelial cell-derived pro-angiogenic chemokine, interleukin-8 (IL-8). These results demonstrate that the up-regulation of bcl-2 expression in tumor-associated endothelial cells enhances intra-tumoral microvascular survival and density and accelerates tumor growth. The ability of bcl-2 to influence the process of capillary network remodeling has also been demonstrated.68 Stable transfection of bcl-2 in bovine aortic endothelial cells (BAEC), which undergo spontaneous involution of the capillary network in vitro, induces inhibition of apoptosis and prevents the capillary network regression. An effect of bcl-2 on the enhancement of maturation of newly formed vessels has also been evidenced.67 In particular, bcl-2 transfection has been found to promote HUVEC survival, to increase the density of the vascular bed and to enhance vascular remodeling, resulting in the formation of mature vascular beds. Remarkably, bcl-2 transfected, but not control HUVEC, recruit an ingrowth of perivascular smooth-muscle alpha-actin-expressing mouse cells, which organize into HUVEC-lined multilayered structures resembling true microvessels.67 As a mechanism by which bcl-2 overexpression in HUVEC results in recruitment of mesenchymal cells and the formation of multilayered vascular structures, the authors suggest that bcl-2, which is not normally expressed by HUVEC, turns on production of a recruitment signal, such as VEGF,69 platelet-derived growth factor,70 angiopoietin-1,71 and/or heparin-binding epidermal growth factor.72 Altogether these data indicate that the

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inhibition of apoptosis in endothelial cells is an essential component of bcl-2 ability to induce angiogenesis. The overexpression of bcl-2 in endothelial cells may protect them from apoptosis induced by inhibitors of angiogenesis resulting in a net gain in new blood vessels, may induce expression of angiogenic factors, may potentiate the ability of endothelial cells to differentiate into functional blood vessels and may enhance the formation of complex vascular structures. As suggested by Nor et al it can be considered a mechanism whereby VEGF-induced expression of bcl-2 may function to enhance the survival of endothelial cells in the toxic, oxygen deficient environment of tumors and ensure the continuous, uninterrupted flow of nutrient to the tumor.61 These results are in agreement with other reports demonstrating that apoptosis can be an essential factor in the inhibition of angiogenesis in vivo. Thrombospondin-1 inhibits angiogenesis in vitro and in vivo by triggering a signaling cascade that culminates in the activation of the apoptotic pathway.64,73 Thrombospondin-1 induces regression of the pre-existing co-opted host vasculature via apoptosis, leading to massive tumor cell loss.65 Inhibition of integrin αvβ3 triggers apoptosis and prevents angiogenesis in an in vivo CAM model,66,68 and results in the dissolution of the tubular network in an in vitro model system of capillary tube formation.68 All these studies raise the possibility that one way endothelial cells “decide” whether or not to initiate and/or to sustain an angiogenic response is by integrating pro- and anti-apoptotic signals, forming new vessels only if survival signals are dominant.64 These data contrast with recent reports showing that inactivation of the apoptotic pathway blocks angiogenesis.65 Recently, Segura et al examined the role of apoptosis in new vessel formation and demonstrated that apoptosis occurs during the angiogenic process in in vitro models, and that it is necessary for correct in vivo remodelling of endothelial cells.65 In particular, inhibition of programmed cells death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis. The authors showed that apoptosis occurs before capillary formation but not after vessels have assembled and that vascular-like structure formation requires apoptotic cell death through activation of a caspase-dependent mechanism and mitochondrial cytochrome c release. Specific inhibition of caspase activation during angiogenesis results in distorted tube-like structures both in vitro and in vivo. Overexpression of human bcl-2 in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel-like structure formation. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis. The evidence that a subset of tumors initially grows by co-opting existing host vessels and that this co-opted host vasculature does not immediately undergo angiogenesis to support the tumor but instead regresses, leading to a secondarily avascular tumor and massive tumor cell loss, is in agreement with these data. Ultimately, however, the remaining tumor is rescued by robust angiogenesis at the tumor margin.62,63 Thus, apoptosis may be a general mechanism in angiogenesis, probably to eliminate superfluous cells not included in the vascular network. The discrepancies observed in the above results can be due, at least in part, to the different phases in which the analyses were performed: very early stages of angiogenesis, when endothelial cell proliferation has not yet begun or is just commencing, may require apoptosis-related phenomena different from those involved in late stage of angiogenesis, when vessels are already formed. While some studies identified mechanisms that operate before vessel formation, other studies analyzed mechanisms involved in vessel maintenance, after angiogenesis is initiated.

Clinical Relevance of VEGF and Bcl-2 Expression Much research has recently focused on the potential clinical relevance of VEGF and bcl-2 expression for evaluating prognosis in individual patients and as therapeutic targets. The first report showing the prognostic value of tumor neovascularization in human breast cancer was published in 1991.74 Since then, intra-tumor microvessel density has been established as an independent prognostic factor in a wide variety of solid tumors (particularly in breast and colon cancers, where it may be of predictive of poor prognosis even in early, node-negative, subsets of patients)75-77 and, more surprisingly, in different hematological

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malignancies.78,79 As the molecular circuitry regulating neo-angiogenesis has become clearer, the focus of research has shifted towards the measure of individual angiogenic factors, most notably VEGF, in an attempt to identify more quantitative and objective parameters to be correlated with clinical outcome. VEGF is overexpressed in nearly all the major solid tumor histological types, as well as in a variety of hematological malignancies.80,81 VEGF overexpression is detected not only in invasive tumors, but also in some premalignant lesions, and its expression levels increase in parallel to malignant progression up to metastatic lesions.82-84 In some (e.g., carcinomas of the breast and cervix), but not all tumor types, VEGF expression is clearly correlated with intra-tumoral microvessel density and with one or more indicators of poor prognosis, including tumor size, metastatic disease, and shorter progression-free and overall survival.80,81 In studies conducted in breast and head and neck cancer, VEGF has been identified as the strongest predictor of survival in multivariate analysis.85,86 Notable exceptions in which such a correlation could not be found include gastric, hepatocellular, and bladder carcinomas.80,81 Although the evidence presented in favor of a prognostic significance of tumor-associated VEGF is compelling, none of techniques used to measure it has been sufficiently validated to warrant its routine use in the clinic. Another possibility is to quantitate circulating VEGF levels. This approach has several potential advantages.80,81 However, the measurement of circulating VEGF levels may be hampered by several confounding factors (see ref. 87 for a comprehensive review). Consequently, great caution should be used when interpreting and comparing the results of different studies. These methodological problems notwithstanding, the bulk of published studies indicate that circulating VEGF levels are consistently higher in cancer patients than in normal subjects and that the amount of circulating VEGF indirectly reflects tumor burden.80,81 Moreover, in most studies high levels of circulating VEGF have been associated with unfavorable clinical parameters, such as disease progression, poor response to chemotherapy, and poor survival. Notable exceptions in which such a correlation could not be found include lung, renal cell, ovarian, and head and neck carcinomas.80,81 In addition to being released in the systemic circulation, VEGF is accumulated at high levels in other body fluids, such as in malignant pleural effusions and ascites, in malignant (as opposed to benign) ovarian cysts,80,81 and in the urine of primary or recurrent superficial bladder cancer patients, where its sensitivity and specificity have been claimed to be superior to urinary cytology.88,89 More recently, several studies have focused on the prognostic role of upstream regulators of VEGF expression, such as members of the HIF family of transcription factors. Although preliminary, the bulk of evidence indicates that HIF-1α and HIF-2α are upregulated in areas of hypoxia in several tumors and that their expression results in the overexpression of the downstream target, VEGF, and in increased microvessel density in several tumor histotypes.36 Overexpression of HIF family members tends to correlate with increasing stage and other unfavorable clinical parameters in colorectal cancer90,91 and has frankly negative prognostic significance for progression-free and overall survival in breast (both lymph node positive and negative subsets),92,93 endometrial,94 cervical,95 and nasopharyngeal carcinomas,96 but not in other cancers (see also ref. 36 for review). Overall, the above-presented evidence suggests that, during their progression towards a more malignant state, many human malignancies do activate an angiogenic program that proceeds from hypoxia to upregulation of HIF transcription factors, expression of VEGF, and increased intra-tumoral microvessel density, in a manner similar to that elucidated by in vitro studies. This angiogenic switch, in turn, confers a more aggressive and invasive phenotype that in most instances results in a poor clinical outcome. The prognostic role of bcl-2 itself and other bcl-2 family members in human malignancies is much more complicated. An accumulating wealth of experimental data, both in vitro and in vivo, indicates that loss of the ability to undergo programmed cell death upon physiological or stress-induced stimulation is intimately related to the ability of cancer cells to progress towards a more malignant and therapy-resistant phenotype.97-99 However, while no doubt exists about the ability of bcl-2 to block apoptosis induced by a wide variety of stimuli in essentially all

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species from worm to man, the prognostic role of its expression in certain human malignancies is sometimes paradoxical.100-103 In fact, while bcl-2 overexpression is unequivocally correlated with resistance to therapy and poor prognosis in leukemias and lymphomas104-109 and in locally advanced or metastatic prostate cancer undergoing hormonal therapy,25,110,111 in other malignancies it has no discernible impact on prognosis or is even associated with better clinical outcome and a more favorable prognosis.102,103,112 This apparently paradoxical role of bcl-2 in tumor progression is particularly striking in breast cancer, where its expression was found to correlate with lower histological tumor grade, normal ploidy, hormone receptor positivity, and absence of metastases, resulting in an overall better survival rate.102,103,112 Although examples of an opposite (i.e., negative) impact of bcl-2 on breast cancer prognosis exist, these findings have sparked an ongoing controversy on the clinical significance of bcl-2 expression in breast and other human cancers (see ref. 113 for a comprehensive review) and have raised the question whether bcl-2 could be a meaningful therapeutic target in these diseases. Detailed discussion of individual studies approaching this question is beyond the scope of this chapter but we will give a brief overview of the working hypotheses that have been formulated to explain this paradox. First, the observation that expression of bcl-2 is estrogen inducible in mammary cells suggests that its presence in breast cancer cells may represent a fortuitous marker of tumors that have arisen from a less aggressive genetic pathway involving a dependence on steroid hormones.112 Second, bcl-2 is a multifunctional protein that not only inhibits apoptotic cell death, but also restrains cell cycle entry and progression.114,115 In breast cancer cells with high bcl-2 expression, this cell cycle-restraining activity may dominant over anti-apoptotic effects, thereby resulting in a better prognosis. Third, more than 20 members of the bcl-2 family have been thus far identified in humans, encompassing pro- and anti-apoptotic proteins. Consequently, bcl-2 expression alone may not be informative, unless it is put in context with the expression of other family members, especially those with pro-apoptotic functions. In agreement with this hypothesis, the ratio of bcl-2 to its pro-apoptotic counterpart Bax is more informative than either member alone in determining the prognosis of acute leukemia patients.105,116 Fourth, bcl-2 family members mainly control the early steps of the apoptotic process. However, loss of downstream positive regulators of the caspase cascade, such as APAF-1 and different caspases, as well as upregulation of downstream caspase inhibitors, such as members of the inhibitor-of-apoptosis-protein (IAP) family, have been described in human tumors.102 Downstream road-blocks in the apoptotic process may bypass the need for bcl-2 expression in order to prevent apoptosis, rendering its function dispensable.117 In this respect, bcl-2 overexpression would paradoxically mark cells that are inherently sensitive to spontaneous or drug-induced apoptosis, whereas bcl-2-negative cells would be intrinsically resistant to the apoptotic process, thereby behaving in a more aggressive and therapy-resistant fashion. Support for this hypothesis comes from clinical data indicating that the prognostic effect of a low Bax/Bcl-2 ratio in acute myeloid leukemia is negative in patients with favorable and intermediate prognosis cytogenetics, but switches to positive in patients with unfavorable cytogenetics, in whom the accumulation of other genetic defects renders the cells inherently resistant to apoptosis.105 Fifth, the evaluation of bcl-2 expression levels for prognostic purposes is usually conducted in bulk tumor populations. Recent data, however, indicate that, within a given tumor, only a minuscule population of cells with stem cell characteristics is able to sustain tumorigenic growth.118 It is reasonable to speculate that analysis of the expression of pro- and anti-apoptotic bcl-2 family members would be more informative if prospectively conducted in this subpopulation of “tumorigenic stem cells”. Finally, an intriguing hypothesis that envisions bcl-2 overexpression as an “anti-progressor” phenotype has been recently formulated by Gurova et al.103 According to their hypothesis, loss of apoptosis through either one of two mutually exclusive routes (bcl-2 overexpression or p53 inactivation) confers a selective growth advantage to cancer cells; however, loss of apoptosis through p53 inactivation also leads to genetic instability and tumor progression, while loss of apoptosis through bcl-2 overexpression creates genetically stable tumors that escape selective pressure to inactivate p53 and are, therefore, less prone to progression, resulting in a better outcome.103

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Figure 1. Schematic diagram showing the involvement of bcl-2 in metastasis and neoangiogenesis. Bcl-2 overexpression enhances metastatic and angiogenic potential of tumor cells by increasing their resistance to apoptosis, by inducing metastasis-associated properties, such as migration and invasion, and by co-operating with hypoxia to induce VEGF and HIF-1α. Bcl-2 overexpression also enhances endothelial cell angiogenesis.

Finally, preclinical evidence is starting to emerge indicating a causal relationship between bcl-2 and VEGF expression in different models of human cancer, but no definitive clinical data are presently available to support or discard such an association. Preliminary reports, however, suggest that the situation in clinical samples could be more complicated than is expected from experimental studies.119,120 Evaluation of the bcl-2/VEGF relationship in adequate clinical series is, therefore, strongly warranted.

Conclusion VEGF is an endothelial and cell-specific mitogen and permeability factor that potently induces angiogenesis. The ability of VEGF to potentiate and sustain angiogenesis is associated with its ability to prolong the survival of endothelial cells and to induce expression of bcl-2. The upregulation of bcl-2 in tumor microvascular endothelial cells not only enhances cell survival, improving the ability of these cells to remain viable and functional despite the constraints imposed by the tumor microenvironment, but also engages them in a more vigorous angiogenic response in vitro and in vivo. Moreover, bcl-2 overexpression increases the angiogenic potential of tumor cells but, in contrast to endothelial cells, this effect seems independent of bcl-2 anti-apoptotic activity. Thus, the regulation of VEGF and angiogenesis is a new function for this oncogene (Fig. 1). From a clinical standpoint, though the role of VEGF and bcl-2 in the prognostic evaluation of individual patient is still a matter of debate, an overwhelming amount of preclinical data, as well as preliminary clinical data,59,121 clearly indicates that both VEGF and bcl-2 are a prom-

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ising therapeutic target. Thus, strategies that block VEGF activity or bcl-2 expression may have multiple anti-tumor effects including induction of apoptosis prevention of endothelial cell growth, and reduction of angiogenic molecules with consequent increase in tumor cell apoptosis leading to an overall reduction in angiogenesis.

Acknowledgments This work was supported by Italian Association for Cancer Research (D.D.B.) and Ministero della Salute (D.D.B.).

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51. Nabors LB, Gillespie GY, Harkins L et al. HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs. Cancer Res 2001; 61:2154-2161. 52. Ricca A, Biroccio A, Del Bufalo D et al. bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells. Int J Cancer 2000; 86:188-196. 53. Huang S, Pettaway CA, Uehara H et al. Blockade of NF-kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion and metastasis. Oncogene 2001; 20:4188-4197. 54. Milella M, Estrov Z, Kornblau SM et al. Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia. Blood 2002; 99:3461-3464. 55. Simoes-Wust AP, Schurpf T, Hall J et al. Bcl-2/bcl-xL bispecific antisense treatment sensitizes breast carcinoma cells to doxorubicin, paclitaxel and cyclophosphamide. Breast Cancer Res Treat 2002; 76:157-166. 56. Jansen B, Zangemeister-Wittke U. Antisense therapy for cancer—The time of truth. Lancet Oncol 2002; 3:672-683. 57. Wang JL, Liu D, Zhang ZJ et al. Structurebased discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. Proc Natl Acad Sci USA 2000; 97:7124-7129. 58. Jansen B, Wacheck V, Heere-Ress E et al. Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet 2000; 356:1728-1733. 59. Genasense; [1 screen]. Available at: URL:http://www.genta.com/genta/Products/genasense.html. Accessed June 4, 2003. 60. Gagnon ML, Bielenberg DR, Gechtman Z et al. Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: In vivo expression and anti-tumor activity. Proc Natl Acad Sci USA 2000; 97:2573-2578. 61. Nor JE, Christensen J, Liu J et al. Up-Regulation of Bcl-2 in microvascular endothelial cells enhances intratumoral angiogenesis and accelerates tumor growth. Cancer Res 2001; 61:2183-2188. 62. Holash J, Maisonpierre PC, Compton D et al. Vessel cooption, regression and growth in tumors mediated by angiopoietins and VEGF. Science 1999; 284:1994-1998. 63. Holash J, Wiegand SJ, Yancopoulos GD. New model of tumor angiogenesis: Dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 1999; 18:5356-5362. 64. Jimenez B, Volpert OV, Crawford SE et al. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 2000; 6:41-48. 65. Segura I, Serrano A, De Buitrago GG et al. Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis. FASEB J 2002; 16:833-841. 66. Brooks PC, Montgomery AM, Rosenfeld M et al. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994; 79:1157-1164. 67. Schechner JS, Nath AK, Zheng L et al. In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse. Proc Natl Acad Sci USA 2000; 97:9191-9196. 68. Pollman MJ, Naumovski L, Gibbons GH. Endothelial cell apoptosis in capillary network remodeling. J Cell Physiol 1999; 178:359-370. 69. Fong GH, Rossant J, Gertsenstein M et al. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 1995; 376:66-70. 70. Hirschi KK, Rohovsky SA, Beck LH et al. Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact. Circ Res 1999; 84:298-305. 71. Suri C, McClain J, Thurston G et al. Increased vascularization in mice overexpressing angiopoietin-1. Science 1998; 282:468-471. 72. Swinscoe JC, Carlson EC. Capillary endothelial cells secrete a heparin-binding mitogen for pericytes. J Cell Sci 1992; 103:453-461. 73. Nor JE, Mitra RS, Sutorik MM et al. Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J Vasc Res 2000; 37:209-218. 74. Weidner N, Semple JP, Welch WR et al. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma. N Engl J Med 1991; 324:1-8. 75. Weidner N. Intratumor microvessel density as a prognostic factor in cancer. Am J Pathol 1995; 147:9-19. 76. Heimann R, Ferguson D, Powers C et al. Angiogenesis as a predictor of long-term survival for patients with node-negative breast cancer. J Natl Cancer Inst 1996; 88:1764-1769.

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77. Takahashi Y, Tucker SL, Kitadai Y et al. Vessel counts and expression of vascular endothelial growth factor as prognostic factors in node-negative colon cancer. Arch Surg 1997; 132:541-546. 78. Padro T, Ruiz S, Bieker R et al. Increased angiogenesis in the bone marrow of patients with acute myeloid leukemia. Blood 2000; 95:2637-2644. 79. Vacca A, Ribatti D, Ruco L et al. Angiogenesis extent and macrophage density increase simultaneously with pathological progression in B-cell non hodgkin’s lymphomas. Br J Cancer 1999; 79:965-970. 80. Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: A critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 2002; 20:4368-4380. 81. Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol 2001; 19:1207-1225. 82. Brown LF, Berse B, Jackman RW et al. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol 1995; 26:86-91. 83. Guidi AJ, Abu-Jawdeh G, Berse B et al. Vascular permeability factor (vascular endothelial growth factor) expression and angiogenesis in cervical neoplasia. J Natl Cancer Inst 1995; 87:1237-1245. 84. Wong MP, Cheung N, Yuen ST et al. Vascular endothelial growth factor is up-regulated in the early pre malignant stage of colorectal tumor progression. Int J Cancer 1999; 81:845-850. 85. Linderholm B, Tavelin B, Grankvist K et al. Vascular endothelial growth factor is of high prognostic value in node- negative breast carcinoma. J Clin Oncol 1998; 16:3121-3128. 86. Smith BD, Smith GL, Carter D et al. Prognostic significance of vascular endothelial growth factor protein levels in oral and oropharyngeal squamous cell carcinoma. J Clin Oncol 2000; 18:2046-2052. 87. Jelkmann W. Pitfalls in the measurement of circulating vascular endothelial growth factor. Clin Chem 2001; 47:617-623. 88. Jones A, Crew J. Vascular endothelial growth factor and its correlation with superficial bladder cancer recurrence rates and stage progression. Urol Clin North Am 2000; 27:191-197. 89. Miyake H, Hara I, Yamanaka K et al. Elevation of serum level of vascular endothelial growth factor as a new predictor of recurrence and disease progression in patients with superficial urothelial cancer. Urology 1999; 53:302-307. 90. Jiang YA, Fan LF, Jiang CQ et al. Expression and significance of PTEN, hypoxia-inducible factor-1 alpha in colorectal adenoma and adenocarcinoma. World J Gastroenterol 2003; 9:491-494. 91. Kuwai T, Kitadai Y, Tanaka S et al. Expression of hypoxia-inducible factor-1alpha is associated with tumor vascularization in human colorectal carcinoma. Int J Cancer 2003; 105:176-181. 92. Bos R, van der GP, Greijer AE et al. Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer 2003; 97:1573-1581. 93. Schindl M, Schoppmann SF, Samonigg H et al. Overexpression of hypoxia-inducible factor 1 alpha is associated with an unfavorable prognosis in lymph node-positive breast cancer. Clin Cancer Res 2002; 8:1831-1837. 94. Sivridis E, Giatromanolaki A, Gatter KC et al. Association of hypoxia-inducible factors 1alpha and 2alpha with activated angiogenic pathways and prognosis in patients with endometrial carcinoma. Cancer 2002; 95:1055-1063. 95. Birner P, Schindl M, Obermair A et al. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 2000; 60:4693-4696. 96. Hui EP, Chan AT, Pezzella F et al. Coexpression of hypoxia-inducible factors 1alpha and 2alpha, carbonic anhydrase IX, and vascular endothelial growth factor in nasopharyngeal carcinoma and relationship to survival. Clin Cancer Res 2002; 8:2595-2604. 97. Igney FH, Krammer PH. Death and anti-death: Tumor resistance to apoptosis. Nat Rev Cancer 2002; 2:277-288. 98. Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol 1999; 17:2941-2953. 99. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: A link between cancer genetics and chemotherapy. Cell 2002; 108:153-164. 100. Baliga BC, Kumar S. Role of Bcl-2 family of proteins in malignancy. Hematol Oncol 2002; 20:63-74. 101. Cory S, Adams JM. The Bcl2 family: Regulators of the cellular life-or-death switch. Nat Rev Cancer 2002; 2:647-656. 102. Blagosklonny MV. Paradox of Bcl-2 (and p53): Why may apoptosis-regulating proteins be irrelevant to cell death? Bioessays 2001; 23:947-953. 103. Gurova KV, Gudkov AV. Paradoxical role of apoptosis in tumor progression. J Cell Biochem 2003; 88:128-137.

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104. Campos L, Rouault JP, Sabido O et al. High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Blood 1993; 81:3091-3096. 105. Kornblau SM, Vu HT, Ruvolo P et al. BAX and PKCalpha modulate the prognostic impact of BCL2 expression in acute myelogenous leukemia. Clin Cancer Res 2000; 6:1401-1409. 106. Wickremasinghe RG, Hoffbrand AV. Biochemical and genetic control of apoptosis: Relevance to normal hematopoiesis and hematological malignancies. Blood 1999; 93:3587-3600. 107. Kitada S, Andersen J, Akar S et al. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: Correlations with In vitro and In vivo chemoresponses. Blood 1998; 91:3379-3389. 108. Prokop A, Wieder T, Sturm I et al. Relapse in childhood acute lymphoblastic leukemia is associated with a decrease of the Bax/Bcl-2 ratio and loss of spontaneous caspase-3 processing in vivo. Leukemia 2000; 14:1606-1613. 109. Stoetzer OJ, Nussler V, Darsow M et al. Association of bcl-2, bax, bcl-xL and interleukin-1 beta-converting enzyme expression with initial response to chemotherapy in acute myeloid leukemia. Leukemia 1996; 10 Suppl 3:S18-S22. 110. Bruckheimer EM, Kyprianou N. Apoptosis in prostate carcinogenesis. A growth regulator and a therapeutic target. Cell Tissue Res 2000; 301:153-162. 111. Colombel M, Symmans F, Gil S et al. Detection of the apoptosis-suppressing oncoprotein bc1-2 in hormone- refractory human prostate cancers. Am J Pathol 1993; 143:390-400. 112. Krajewski S, Krajewska M, Turner BC et al. Prognostic significance of apoptosis regulators in breast cancer. Endocr Relat Cancer 1999; 6:29-40. 113. Hamilton A, Piccart M. The contribution of molecular markers to the prediction of response in the treatment of breast cancer: A review of the literature on HER-2, p53 and BCL-2. Ann Oncol 2000; 11:647-663. 114. Huang DC, O’Reilly LA, Strasser A et al. The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry. EMBO J 1997; 16:4628-4638. 115. Konopleva M, Tari AM, Estrov Z et al. Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other anti-apoptotic proteins. Blood 2000; 95:3929-3938. 116. Del Poeta G, Venditti A, Del Principe MI et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood 2003; 101:2125-2131. 117. Vilenchik M, Raffo AJ. Benimetskaya L et al. Antisense RNA down-regulation of bcl-xL Expression in prostate cancer cells leads to diminished rates of cellular proliferation and resistance to cytotoxic chemotherapeutic agents. Cancer Res 2002; 62:2175-2183. 118. Al Hajj M, Wicha MS, Benito-Hernandez A et al. From the Cover: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100:3983-3988. 119. Bairey O, Zimra Y, Shaklai M et al. Bcl-2 expression correlates positively with serum basic fibroblast growth factor (bFGF) and negatively with cellular vascular endothelial growth factor (VEGF) in patients with chronic lymphocytic leukemia. Br J Haematol 2001; 113:400-406. 120. Koukourakis MI, Giatromanolaki A, O’Byrne KJ et al. bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in nonsmall-cell lung cancer. Clin Exp Metastasis 1999; 17:545-554. 121. Rosen LS. Clinical experience with angiogenesis signaling inhibitors: Focus on vascular endothelial growth factor (VEGF) blockers. Cancer Control 2002;9 (2 Suppl):36-44.

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CHAPTER 5

Vascular Endothelial Growth Factor in Breast Cancer Tilmann Lantzsch and Lukas Hefler

Abstract

B

reast cancer, as with most solid tumors, needs to develop the angiogenic phenotype for invasiveness, progression and metastasis. Several studies have determined that the degree of peritumoral vascularity as a marker of angiogenesis is associated with prognosis of patients operated on for early stage invasive breast cancer. Angiogenesis is regulated by a network of inducing and inhibiting factors under physiologic conditions, whereas in pathologic conditions, such regulation is altered or absent. These regulating factors include cytokines, fibrin, and integrins, among others. A number of pro-angiogenic factors have been identified, the most potent of which are vascular endothelial growth factor/ vascular permeability factor (VEGF/VPF), acidic and basic fibroblast growth factor (aFGF and bFGF) and epidermal growth factor (EGF). Vascular endothelial growth factor (VEGF) has been shown to play a major role in tumor angiogenesis and is a marker of tumor outcome and prognosis in breast cancer. In conclusion angiogenesis will be important in evaluating the risk of metastasis in breast cancer whereas angiogenesis inhibition may be important in the development of new strategies in cancer therapy.

Introduction In the last century breast and lung cancer were the most important causes of cancer death in women. Breast cancer mortality has not changed for the last 50 years. In recent years there has been a slight overall decline in breast-cancer age-adjusted mortality. Nearly all of the reduction in mortality is accounted for by early diagnosis of in-situ or invasive lesions of less than 2 cm with screening mammography. Reduced mortality might be a reflection of earlier diagnosis and more effective treatment, including adjuvant systemic therapy. There are some well documented risk factors for breast cancer in women: age over 50 years, family history of breast cancer, higher socioeconomic status, nulliparous, or the first full term pregnancy after age 30.1It was shown that breast cancer is more frequent in Jewish women compared with non-Jewish women and in black women compared with white women. Low incidence and mortality rates for female breast cancer are found in most Asian and African countries, and high rates in North America and northern European countries.2 Several large-scale studies have failed to demonstrate a correlation between the prolonged use of oral contraceptives and breast cancer.3,4 In an analysis of 961 women born in 1944 or later who took oral contraceptives during their whole reproductive span no increase in breast-cancer incidence was reported. Stanford et al found no increased breast cancer risk in middle-aged women taking estrogen and progestin as hormone replacement therapy in a case-control study of 537 women with breast cancer and 492 control women without breast VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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cancer.5 Long-term use (eight years and more) of the combined estrogen and progestin hormone-replacement therapy regimen was associated with a reduced risk of breast cancer.5On the other hand, Colditz and coworkers found an increased risk of breast cancer in postmenopausal patients with combined estrogen and progestin therapy compared with postmenopausal women who had never used hormones.6 New results on breast cancer and hormone replacement therapy were published in the million women study.7 The study described the risk of breast cancer after hormone replacement therapy in 1,084,110 British women. The group found that current use of hormone replacement therapy is associated with increased risk of incident and fatal breast cancer. Furthermore, they showed a substantially higher risk for breast cancer in women with estrogen-progestagen combinations than other types of hormone replacement therapy.

VEGF and Tumor Growth Angiogenesis and metastasis have been the focus of intense cancer research over the past decade. Angiogenesis is well recognized as an essential process for tumor growth, invasion and metastastic spread in many human malignancies.8 New anti-cancer therapies are integrated as the result of understanding the important role of angiogenesis and metastasis in malignant cell dissemination. The formation of new blood vessels from a pre-existing vascular network, has been shown to be essential to tumor growth and survival.9 The extent of neovascularization of tumors correlates well with clinical outcome.10Early mouse experiments established that angiogenesis is required for tumor expansion. The transition from in situ carcinoma to invasive cancer was shown to be preceded by neovascularization.11,12 Tumor invasion is limited by nutrient requirements.13,14,15 The formation of new vessels begins with activation of normally homeostatic endothelial cells to create vascular sprouts that elongate and invade into local stroma toward the tumor. These vessels connect with existing vascular networks as functional capillaries and play an important role in tumor aggressiveness and prognosis.16 Angiogenesis is regulated by a network of inducing and inhibiting factors under physiologic conditions, whereas in pathologic conditions, such regulation is altered or absent. These factors include cytokines, fibrin, and integrins, among others.17-20 Other promoters of angiogenesis have been identified, among the most potent of which are vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), acidic and basic fibroblast growth factor (aFGF and bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), hepatocyte growth factor(HGF), transforming growth factor α and β (TGF α and β), angiotropin, angiogenin, and tumor necrosis factor α (TNFα).18,21 Vascular endothelial growth factor (VEGF) has been shown to play a major role in tumor angiogenesis in many studies.22,23 Breast cancer has served as a paradigm for understanding the biology of angiogenesis and its effects on tumor outcome and patient prognosis. The first study to examine intra-tumoral microvessel density (MVD) as a morphological criteria to identify vessels in histological sections of tumors was carried out by Weidner et al in 1991 using an antibody against factor-8 related antigen as an endothelial marker.24 This work was perfomed on a series of 49 breast carcinomas and demonstrated a near linear relationship between increased microvessel counts and metastasis in breast cancer. In a review of the literature Gasparini described the prognostic role of angiogenesis in breast cancer.25 Most of the reviewed studies found that increased microvessel counts were predictive of worse outcome, although this was not universally confirmed as several studies found no association with prognosis. These discrepancies have been variously explained by factors such as low patient numbers, nonconsecutive series or methodological difficulties using factor 8 related antigen as the endothelial marker which is now generally regarded to be less reliable than other markers such as CD34 or CD31. The crucial role of angiogenesis in breast cancer and its correlation with metastatic disease has been clearly demonstrated.26-29 Previously published studies used various markers

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and techniques to assess the angiogenic potential of breast cancer tissue.26,30-33 Data are conflicting with respect to which marker is most reliable in reflecting angiogenic processes.26,27,30,33 Most commonly, angiogenesis in breast cancer has been assessed by evaluating intra-tumoral microvessel density (MVD), immunohistochemically detected VEGF, tumor tissue VEGF and circulating VEGF concentrations.24,26-28,30 VEGF, which is also known as Vascular Permeability Factor, is widely recognized as the most promising marker of angiogenesis. VEGF stimulates angiogenesis by increasing vascular permeability and by acting as an endothelial cell mitogen by binding to its receptors, VEGFR-1 (VEGF receptor 1 also known as Flt-1, fms-like tyrosine kinase) and VEGFR-2 (KDR/Flk-1, Fetal liver kinase 1 is the murine homologue of human Kinase insert Domain-containing Receptor).34-37 The clinical value of angiogenic parameters including VEGF has been debated. However, a number of studies reported angiogenic factors to be independent prognostic markers and predictors of lymph node involvement.38-40 Furthermore, it has been suggested that serial serum VEGF measurements might predict the optimal time of breast cancer surgery.41 It was found that serum VEGF levels were elevated in patients with invasive cancer of ductal/no specific type, ductal carcinoma in situ and estrogen receptor positive tumors. Patients with lobular carcinoma and estrogen receptor negative tumors had decreased serum VEGF levels comparable with those in the control group.41Recently, serum VEGF levels have also been suggested to serve as monitoring markers in chemotherapeutic trials involving taxanes but others, however, refuted these theories.42,43 Serum VEGF has also been investigated in other human malignancies, including colorectal, vulvar, and ovarian cancer.44-46 Previously published data with respect to serum VEGF in breast cancer are contradictory. It was found that serum VEGF was indicative of tumor bulk, as already known for other malignancies including melanoma and colorectal cancer.47,48 Furthermore, serum VEGF seems to parallel angiogenic processes in the development of lymphatic spread. This is consistent with the concept of angiogenesis as promoter of malignant growth. Although it is reasonable to speculate that elevated serum VEGF was due to tumor cell production, it has to be stated that other possible sources of serum VEGF have been described, e.g., platelets during platelet aggregation, activated human neutrophils, T lymphocytes, and blood mononuclear cells.49-51 Serum VEGF is now known to be largely platelet-derived and plasma VEGF is now considered a more accurate measure of circulating VEGF and any disease related overspill.27

Serum VEGF and Breast Cancer It was hypothesized that intra-tumoral expression of angiogenic proteins correlates with clinical and biological features of breast cancer and serum VEGF, as a circulating surrogate marker of angiogenesis. We previously investigated a panel of immunohistochemical markers eg. intra-tumoral microvessel density, vascular endothelial growth factor and its receptor VEGFR-2/Flk-1, and serum VEGF in 46 patients with breast cancer.52 VEGF and VEGFR-2/ Flk-1, however, fell short of showing any correlation with clinicopathologic parameters. Of note, no correlation was ascertained between intra-tumoral VEGF and serum VEGF. This might be attributed to the fact that local, i.e., intra-tumoral, over-expression of VEGF is not sufficient by itself to cause changes in serum VEGF levels. However, Adams et al reported that plasma VEGF in patients with localized breast cancer was higher than controls or patients with benign breast disease.27 Increased intra-tumoral MVD has been proposed to be the most accurate marker of angiogenesis reflecting enhanced tumor growth and tumor cell de-differentiation.53 Supporting this view, we showed a significant correlation between increased MVD and advanced tumor stage and high tumor grade. Furthermore, a significant correlation between intra-tumoral MVD and serum VEGF was shown.52 Although no direct link between local and systemic processes of angiogenesis could be established, our results are highly indicative of breast cancer as the primary source of the observed elevated serum VEGF.

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A shortcoming of our study was that we did not evaluate the impact of serum VEGF and the investigated immunohistochemical markers on patient prognosis. Of note, the aim of our study was to determine the correlation between intra-tumoral angiogenic proteins with clinicopathological parameters of the disease and the most promising circulating surrogate marker of angiogenesis, i.e., VEGF. The influence of serum VEGF prior to therapy on survival has been previously published.38 But in a critical analysis Gasparini reviewed the studies on circulating VEGF in cancer patients and concluded that there is an unacceptable variability of concentrations even among healthy control subjects from different studies.54 Another controversial point is the definition of the cut-off level of concentration of serum VEGF to classify a patient with high or low levels of the factor. In conclusion the interpretation of circulating VEGF as a prognostic marker is difficult and not clearly defined.

VEGF and Clinical Consequences To analyze the clinical consequences of intra-tumoral VEGF in breast cancer patients, numerous studies were initiated in the last years. Stacker et al55 found an important influence of VEGF-C and VEGF-D in lymphangiogenesis. VEGF-C and VEGF-D are ligands for VEGFR-3/ Flt-4. It was found that VEGFR-3/Flt-4 is predominantly expressed on lymphatic endothelium in normal adult tissues; it is also up-regulated on blood vessel endothelium in tumors and in wound healing.56-59 It was shown that VEGF-C and VEGF-D promoted the development of only tumor-associated peri-tumoral lymphatics and this correlated with an increased rate of metastatic spread.60 In a mouse model it was demonstrated that potential inhibitors of the VEGFR-3 lymphangiogenetic signaling pathway include monoclonal antibodies (mAbs) that block the binding of VEGF-C and VEGF-D to VEGFR-3. A neutralizing VEGF-D mAb that blocked binding to both VEGFR-2 and VEGFR-3 inhibited angiogenesis, lymphangiogenesis, and metastatic spread via the lymphatics in a mouse tumor model that secreted recombinant VEGF-D.61 Nakamura et al described the prognostic significance of VEGF-D in breast carcinoma with long-term follow-up and found a significant correlation between elevated VEGF-D expression, lymph node metastasis and high c-erbB-2 (Her2neu/Her2) expression.62 Elevated VEGF-D was also associated with reduced disease-free survival and overall survival. It was concluded that VEGF-D may be useful in the treatment of breast cancer as a decision-making biomarker for aggressive treatment after operation. Another study described the inhibition of VEGFR-2 with a monoclonal antibody 2C3 that blocks VEGF binding.63 The authors described the therapeutic effects of 2C3 on tumor growth in an orthotopic model of human breast carcinoma implanted in the mammary fat pads of nude mice. They found an inhibition of tumor growth by 75% and a decreased metastatic rate. Another study determined VEGF-C protein expression in a series of breast carcinomas and correlated this with axillary lymph node metastases, the presence of lympho-vascular invasion, bone marrow micro-metastases and other clinicopathological data including estrogen receptor and c-erbB-2 status.64 These authors found an association between VEGF-C expression and c-erbB-2. This correlation suggests a functional relationship and may explain the aggressive phenotype associated with c-erbB-2-positive tumors. In constrast to other carcinomas they did not find an association between VEGF-C expression and regional lymph node metastases in breast cancer.65-67 The inhibition of lymphangiogenesis or angiogenesis seems to be a potential new target for development of anti-cancer therapeutics. Furthermore, breast cancer tissue is highly responsive to changes in ovarian hormone concentrations that induce a cyclic remodelling process involving epithelial, stromal and vascular components during each menstrual cycle.68 In particular, the angiogenic turnover is regulated by estrogens and progesterone, which modulate the expression of VEGF in the epithelial cells of the terminal ductal-lobular units.69 Coradini et al evaluated 212 postmenopausal women with resectable node-positive estrogen receptor-positive breast cancer who received adjuvant tamoxifen.70 The study investigated the comprehensive effect on relapse free survival of VEGF content and steroid receptor profile, evaluated in the same cytosolic fraction. In patients with

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high level of VEGF and low estrogen receptor concentration, they found that tamoxifen failed to impact on the tumors metastatic potential and more tailored adjuvant treatment would be required including, for example, an anti-angiogenic agent. McNamara et al demonstrated that tamoxifen inhibits VEGF-mediated angiogenesis in the rat.71Furthermore, they found that tamoxifen reduces proliferation of a VEGF-dependent endothelial cell line in vitro. This study indicated that tamoxifen may directly inhibit the effect of VEGF on endothelial cell proliferation. Takei et al showed that tamoxifen inhibited estradiol-induced VEGF expression in estrogen-receptor positive breast cancer cell lines (MCF-7 cells).72 On the other hand Berns et al described that combination of p53 gene mutation and high levels of cytosolic VEGF in estrogen receptor (ER)-α-positive primary breast tumors predict a poor outcome for patients treated with first line tamoxifen for advanced disease.73 These patients might benefit more from other types of treatment protocols,eg., systemic chemotherapy.

Future Strategies in Breast Cancer A new study by Connolly et al investigated the effect of selective and nonselective cyclooxygenase inhibition on tumor growth and metastasis in an orthotopic model of breast cancer.74 Cyclooxygenase activity is implicated in the development and growth of malignant tumors.75 Connolly et al injected mammary adenocarcinoma cells into the mammary fat pad of female BALB/c mice. They found a significantly reduced tumor diameter in animals treated with the selective cyclooxygenase 2 (COX-2) inhibitor, SC-236, relative to controls. The number of lung metastases and the microvessel densitry were also reduced in this group. In conclusion, the selective cyclooxygenase inhibitors decreased VEGF production and may be of value in the treatment of primary and metastatic breast cancer. A future strategy for cancer therapy is the blockade of signaling molecules playing a key role in cell proliferation, angiogenesis, and apoptosis. The epidermal growth factor receptor (EGFR) and the c-AMP-dependent protein kinase A have recently been recognized as being among the potentially relevant therapeutic targets.76,77 A new oral active, selective EGFR tyrosine kinase inhibitor called ZD1839 (Iressa, Astra Zeneca) that blocks signal transduction pathways of cancer cells is under clinical development. Tortora et al demonstrated an anti-angiogenic effect on human cancer growth and angiogenesis when the COX-2 inhibitor SC-236 was combined with the selective EGFR tyrosine kinase inhibitor ZD 1839 (Iressa) and the DNA/RNA-mixed backbone oligonucleotide antisense-protein kinase A type I (AS-PKAI).78They measured VEGF, bFGF and vessel formation after treatment and found potent anti-tumor and anti-angiogenic activity after oral administration of these agents in mice. Future studies will show the clinical importance of cyclooxygenase inhibition. In conclusion angiogenesis will be important in evaluating the risk of metastasis in breast cancer whereas angiogenesis inhibition may be important in the development of new strategies in cancer therapy.

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9. Folkman J. Clinical application of research on angiogenesis. N Engl J Med 1995; 333:1757-1763. 10. Weidner N, Folkman J. Tumoral vascularity as a prognostic factor in cancer. Important Adv Oncol 1996; 3:167-190. 11. Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992; 3:65-71. 12. Folkman J, Hochberg M, Knighton D. Self-regulation of growth in three dimensions: The role of surface area limitations. In: Clarkson B, Baserga R, eds. Control of proliferation in animal cells. Cold Spring Harbor Conference on Cell Proliferation 1. Cold Spring Harbor Laboratory Press, 1974; 833. 13. Holmgren L, O’Reilly MS, Folkman J. Dormancy of micrometastases: Balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1995; 1:149-153. 14. Liotta LA, Kleinerman J, Saidel G. Quantitative relationships of intravascular tumor cells: Tumor vessels and pulmonary metastases following tumor implantation. Cancer Res 1974; 34:997-1004. 15. Parangi S, O’Reilly M, Christofori G et al. Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc Natl Acad Sci USA 1996; 93:2002-2007. 16. Brown MR, Masiero L, Kohn EC. Tumor Angiogenesis and Metastasis. In: Hoskins WJ, Perez CA, Young RC eds. Principles and Practice of Gynecologic Oncology. 3 rd ed. Lippincott Williams and Wilkins, 2000; 87-101. 17. Dvorak HF. Tumors: Wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 1986; 315:1650-1659. 18. Folkman J, Klagsbrun M. Angiogenic factors. Science 1987; 235:442-447. 19. Ingber DE, Folkman J. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: Role of extracellular matrix. J Cell Biol 1989; 109:317-330. 20. O’Reilly MS, Holmgren L, Shing Y et al. Angiostatin. A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79:315-328. 21. Joseph-Silverstein J. Silverstein RL. Cell adhesion molecules: An overview Cancer Invest 1998; 16:176-182. 22. Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis. Rec Prog Horm Res 2000; 55:15-36. 23. Tou M, Mastsumoto T, Bando H. Vascular endothelial growth factor : Its prognostic, predictive, and therapeutic implications. Lancet Oncol 2001; 2:667-673. 24. Weidner N, Semple JP, Welch WR et al. Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med 1991; 324:1-8. 25. Gasparini G. Prognostic and predictive value of intra-tumoral microvessel density in human solid tumors. In: Bicknell R, Lewis CE, Ferrara N, eds. Tumor angiogenesis. Oxford: Oxford University Press, 1997; 29-44. 26. Obermair A, Kucera E, Mayerhofer K et al. Vascular Endothelial Growth Factor (VEGF) in Human Breast Cancer: Correlation With Disease-free Survival. Int J Cancer 1997; 74:455-458. 27. Adams J, Carder PJ, Downey S et al. Vascular endothelial growth factor (VEGF) in breast cancer: Comparison of plasma, serum, and tissue VEGF and microvessel density and effects of tamoxifen. Cancer Res 2000; 60:2898-2905. 28. Harmey J. Elevated vascular endothelial growth factor in breast cancer. Eur J Cancer 1996 ; 32:19. 29. Saaristo A, Karpanen T, Alitalo K: Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis. Oncogene 2000; 19:6122-6129. 30. Callagy G, Dimitriadis E, Harmey J et al. Immunohistochemical measurement of tumor vascular endothelial growth factor in breast cancer. A more reliable predictor of tumor stage than microvessel density or serum vascular endothelial growth factor. Appl Immunohistochem Molecul Morphol 2000; 8:104-109. 31. Balsari A, Maier JA, Colnaghi MI et al. Correlation between tumor vascularity, vascular endothelial growth factor production by tumor cells, serum vascular endothelial growth factor levels, and serum angiogenic activity in patients with breast carcinoma. Lab Invest 1999; 79:897-902. 32. Salven P, Perhoniemi V, Tykka H et al. Serum VEGF levels in women with a benign breast tumor or breast cancer. Breast Cancer Res Treat 1999; 53:161-166. 33. Locopo N, Fanelli M, Gasparini G. Clinical significance of angiogenic factors in breast cancer. Breast Cancer Res Treat 1998; 52:159-173. 34. Senger D, Galli S, Dvorak A et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983; 219:983-985. 35. Ferrara N, Heinzel W. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989; 161:851-859. 36. Neufeld G, Cohen T, Gitay-Goren H et al. Similarities and differences between the vascular endothelial growth factor (VEGF) splice variants. Cancer Metas Rev 1996; 15:153-158.

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37. Terman B, Dougher-Vermazen M. Biological properties of VEGF/VPF receptors. Cancer Metast Rev 1996; 15:159-163. 38. Gasparini G, Toi M, Gion M et al. Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma. J Natl Cancer Inst 1997; 89:139-147. 39. Linderholm BK, Lindahl T, Holmberg L et al. The expression of vascular endothelial growth factor correlates with mutant p53 and poor prognosis in human breast cancer. Cancer Res 2001; 61:2256-2260. 40. Skobe M, Hawighorst T, Jackson DG et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001; 7:192-198. 41. Heer K, Kumar H, Speirs V et al. Vascular endothelial growth factor in premenopausal women— indicator of the best time for breast cancer surgery? Br J Cancer 1998; 78:1203-1207. 42. Lissoni P, Fugamalli E, Malugani F et al. Chemotherapy and angiogenesis in advanced cancer: vascular endothelial growth factor (VEGF) decline as predictor of disease control during taxol therapy in metastatic breast cancer. Int J Biol Markers 2000; 15:308-311. 43. Byrne GJ, Bundred NJ. Surrogate markers of tumoral angiogenesis. Int J Biol Markers 2000; 15:334-339. 44. Fujisaki K, Mitsuyama K, Toyonaga A et al. Circulating vascular endothelial growth factor in patients with colorectal cancer. Am J Gastroenterol 1998; 93:249-252. 45. Hefler L, Tempfer C, Obermair A et al. Serum concentrations of vascular endothelial growth factor in vulvar cancer. Clin Cancer Res 1999; 5:2806-2809. 46. Tempfer C, Obermair A, Hefler L et al. Vascular endothelial growth factor serum concentrations in ovarian cancer. Obstet Gynecol 1998; 92:360-363. 47. Ugurel S, Rappl G, Tilgen W et al. Increased serum concentration of angiogenic factors in malignant melanoma patients correlates with tumor progression and survival. J Clin Oncol 2001; 19:577-583. 48. Takeda A, Shimada H, Imaseki H et al. Clinical significance of serum vascular endothelial growth factor in colorectal cancer patients: Correlation with clinicopathological factors and tumor markers. Oncol Rep 2000; 7:333-338. 49. Verheul H, Hoekman K, Luykx-de Bakker S et al. Platelet: Transporter of vascular endothelial growth factor. Clin Cancer Res 1997; 3:2187-2190. 50. Maloney J, Silliman C, Ambruso D et al. In vitro release of vascular endothelial growth factor during platelet aggregation. Am J Physiol 1998; 275:1054-1061. 51. Webb N, Myers C, Watson C et al. Activated human neutrophils express vascular endothelial growth factor (VEGF). Cytokine 1998; 10:254-257. 52. Lantzsch T, Hefler L, Krause U et al. The correlation between immunohistochemically-detected markers of angiogenesis and serum vascular endothelial growth factor in patients with breast cancer. Anticancer Res 2002; 22:1925-1928. 53. Abulafia O, Triest WE, Sherer DM. Angiogenesis in malignancies of the female genital tract. Gynecol Oncol 1999; 72:220-231. 54. Gasparini G. Clinical significance of determination of surrogate markers of angiogenesis in breast cancer. Crit Rev Oncol Hematol 2001; 37:97-114. 55. Stacker SA, Baldwin ME, Achen MG. The role of tumor lymphangiogenesis in metastastic spread. FASEB J 2002; 16:922-934. 56. Kaipainen A, Korhonen J, Mustonen T et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci 1995; 92:3566-3570. 57. Kukk E, Lymboussaki A, Taira S et al. VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 1996; 122:3829-3837. 58. Partanen TA, Alitalo K, Miettinen M. Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors. Cancer 1999; 86:2406-2412. 59. Paavonen K, Poulakkainen P, Jussila L et al. Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am J Pathol 2000; 156:1499-1504. 60. Kinoshita J, Kitamura K, Kabashima A et al. Clinical significance of vascular endothelial growth factor-C (VEGF-C) in breast cancer. Breast Cancer Res Treat 2001; 66:159-164. 61. Stacker SA, Caesar C, Baldwin ME et al. VEGF-D promotes the metastasic spread of tumor cells via the lymphatics. Nature Med 2001; 7:186-191. 62. Nakamura Y, Yasuoka H, Tsujimoto M et al. Prognostic significance of vascular endothelial growth factor D in breast carcinoma with long-term follow-up. Clin Cancer Res 2003; 9:716-721. 63. Zhang W, Ran S, Sambade M et al. A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model. Angiogenesis 2002; 5:35-44.

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64. Hoar FJ, Chaudhri S, Wadley MS et al. Coexpression of vascular growth factor C (VEGF-C) and c-erbB-2 in human breast carcinoma. Eur J Cancer 2003; 39:1698-1703. 65. Yonemura Y, Endo Y, Fujita H. Role of vascular endothelial growth factor C expression in the development of lymph node metastasis in gastric cancer. Clin Cancer Res 1999; 5:1823-1829. 66. Akagi K, Ikeda Y, Miyazaki M. Vascular endothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues. Br J Cancer 2000; 83:877-891. 67. Ueda M, Terai Y, Yamashita Y. Correlation between vascular endothelial growth factor-C expression and invasion phenotype in cervical carcinomas. Int J Cancer 2002; 98:335-343. 68. Vogel PM, Georgiade NG, Fetter BF et al. The correlation of histologic changes in human breast with the menstrual cycle. Am J Pathol 1981; 104:23-34. 69. Nakamura J, Lu Q, Aberdeen G et al. The effect of estrogen on aromatase and vascular endothelial growth factor messenger ribonucleic acid in the normal nonhuman primate mammary gland. J Clin Endocrinol Metab 1999; 84:1432-1437. 70. Coradini D, Biganzli E, Pellizaro C et al. Vascular endothelial growth factor in node-positive breast cancer patients treated with adjuvant tamoxifen. Br J Cancer 2003; 89:268-270. 71. McNamara DA, Harmey J, Wang JH et al. Tamoxifen inhibits endothelial cell proliferation and attenuates VEGF-mediated angiogenesis and migration in vivo. Eur J Surg Oncol 2001; 27:714-718. 72. Takai H, Lee ES, Jordan VC. In vitro regulation of vascular endothelial growth factor by estrogens and anitestrogens in estrogen-receptor positive breast cancer. Breast Cancer 2002; 9:39-42. 73. Berns EM, Klijn JGM, Look MP et al. Combined vascular endothelial growth factor and tp53 status predicts poor response to tamoxifen therapy in estrogen receptor-positive advanced breast cancer. Clin Cancer Res 2003; 9:1253-1258. 74. Connolly EM, Harmey JH, O’Grady T et al. Cyclo-oxygenase inhibition reduced tumor growth and metastasis in an orthotopic model of breast cancer. B J Cancer 2002; 87:231-237. 75. Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (part II). J Natl Cancer Inst 1998; 90:1609-1620. 76. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: Targeting the epidermal growth factor receptor. Clin Cancer Res 2001; 7:2958-2970. 77. Cho-Chung YS, Pepe S, Clair T et al. cAMP-dependent protein kinase: Role in normal and malignant growth. Crit Rev Oncol Hematol 1995; 21:33-61. 78. Tortora G, Caputo R, Damino V et al. Combination of a selective cyclooxygenase-2 inhibitor with epidermal growth factor receptor tyrosine kinase inhibitor zd1839 and protein kinase a antisense causes cooperative antitumor and antiangiogenic effect. Clin Cancer Res 2003; 9:1566-1572.

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CHAPTER 6

VEGF and Tumor Progression in Human Melanoma Domenico Ribatti, Angelo Vacca and Franco Dammacco

Abstract

A

ngiogenesis occurs in pathological conditions, such as tumors, where a specific critical point is the transition from the avascular to the vascular phase. Tumor angiogenesis depends mainly on the release by neoplastic cells of growth factors specific for endothelial cells, able to stimulate the growth of the host’s blood vessels. This article summarises the literature concerning the relationship between angiogenesis and progression in human melanoma and the specific role played by vascular endothelial growth factor (VEGF). The recent applications of anti-angiogenic agents which interfere with VEGF signalling and block melanoma progression, are also described.

Introduction Tumor angiogenesis is regulated by several factors, mainly growth factors for the endothelial cells secreted by the tumor and host inflammatory cells, and mobilized from extracellular matrix stores by proteases secreted by tumor cells.1 The extracellular matrix components and endothelial cell integrins, hypoxia, oncogenes and tumor suppressor genes are other regulatory factors. Angiogenesis is mandatory for tumor progression in terms of growth, invasion and metastasis since it supplies oxygen and metabolites, whereas endothelial cells secrete growth factors for tumor cells and a variety of proteinases which facilitate their invasion and entry into the circulation. Angiogenic factors are potent growth factors that promote proliferation and migration of endothelial cells. Several have been identified, including vascular endothelial growth factor (VEGF), placental growth factor (PlGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8) and angiopoietins-1 and –2.1 VEGF is an angiogenic factor in vivo and in vitro with effects on vascular permeability.2 VEGF and VEGF receptors (VEGF-Rs) play a critical role in the control of blood vessels because they are the first endothelial cell-specific signal transduction pathways activated during vascular development and are critical molecules in this process, as evidenced in embryos homozygous for a targeted null mutation in their genes.3

VEGF in Tumor Angiogenesis VEGF is expressed in most solid tumors and the VEGF-Rs are predominant in endothelial cells surrounding or penetrating malignant tissue, but are absent from vascular cells in the surrounding normal tissue.4 This finding suggests that VEGF-R expression is induced in endothelial cells during tumor angiogenesis by VEGF secreted by tumor cells. VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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The amount of VEGF expressed by cancer cells correlates with poor prognosis in many types of tumors, including carcinoma of the breast, kidney, colon, brain, ovary, cervix, thyroid, bladder, esophagus, and prostate and in osteoid and soft tissue sarcomas and pediatric tumors.2 Anti-VEGF blocking antibodies, antisense VEGF cDNA and a dominant negative VEGFR mutant inhibit tumor growth in different experimental models.5-7 There is strong evidence that antibodies that neutralize VEGF and antibodies that actually block VEGF-R retard tumor growth and may reduce tumor size in mice, effects that are mediated through inhibition of angiogenesis.2 Antibodies that selectively recognize the complex that VEGF forms with its receptor 2 (VEGFR-2 also known as KDR/Flk-1 where Flk-1 is murine homologue of human KDR) on vascular endothelium have also been developed.8

Angiogenesis and Human Melanoma Human malignant melanoma is a highly metastatic tumor of increasing incidence, poor prognosis, and high resistance to treatment, especially when it hass metastasized. It progresses through a series of steps: nevocellular nevi, dysplastic nevi, in situ melanoma, radial growth phase melanoma (Breslow index < 0.75 mm), vertical growth phase melanoma (Breslow index > 0.75 mm) and metastatic melanoma.9 As it progresses, melanoma acquires a rich vascular network. It was initially suggested that this increased tumor vascularity, as determined by percentage of vascular area at the tumor base, was of prognostic significance in lesions of intermediate thickness (0.76 to 4.0 mm).10,11 In one study, 20 such melanomas (10 with metastasis and 10 without) were analyzed using Ulex europaeus I agglutinin staining, specific for endothelial cells. The percentage of vascular area at the base of the tumor was more than two-fold higher in the recurrence group than in the relapse-free group.10 In a study of 71 melanomas, using Doppler ultrasonography and Ulex staining, these authors later demonstrated that the neovascular bed increases as tumor thickness approaches 0.8 mm.11 These results were subsequently confirmed by others and it was further demonstrated that an increasing proportion of melanoma tumor cells express the laminin receptor as tumor thickness increases which enables their adhesion to the vascular wall.12-14 In 1994, the extent of vascularization, expressed as mean vessel number, was found to be predictive of metastasis and death in thin (< 0.75 mm) melanomas.15 Straume et al examined the microvessel density of 88 nodular melanomas using factor-VIII related antigen and demonstrated that increased microvessel density was associated with reduced overall survival.16 Interestingly, they found a correlation between vessel density and ulceration in this subgroup. Massi et al have since suggested that neovascularization is a critical event in the progression of thick (> 3 mm) melanoma.17 It has also been reported that angiogenesis may have a key role in the metastasis of thin melanomas exhibiting extensive histopathological regression.18 These authors showed that the mean microvessel counts of thin regressing melanomas were higher than those of melanomas without regression, especially those of radial growth phase melanomas. Other studies, however, have provided evidence against the prognostic value of tumor vascularity in cutaneous melanoma.19,20 Busam et al performed a matched-pair study of 60 cases of metastasizing and nonmetastasizing melanomas matched for tumor thickness, age, sex and anatomic size and found no significant differences in the microvessel density between the two groups.19 In melanoma several angiogenic cytokines are expressed, including bFGF, IL-3 and IL-8, VEGF and granulocyte macrophage colony stimulating factor (GM-CSF).21-26 Blood values of bFGF, VEGF and IL-8 were positively correlated with disease stage and tumor burden, and implied poor overall survival and high probability of progression.24 bFGF is apparently the most important growth factor in melanoma. Reed et al detected bFGF mRNA in tumor cells of metastatic and primary invasive melanoma, but not in melanoma in situ, nor in benign melanocytic nevi.27 The metastatic potential and invasiveness of human melanoma cells are also markedly increased on transduction with the bFGF gene.22

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Inhibition of bFGF production by antisense oligodeoxynucleotides led to inhibition of melanoma proliferation in vitro and in vivo.28,29 IL-8 was identified as essential an autocrine growth factor for melanoma cells, and a direct correlation between its level of expression in cell lines of different metastatic potential was observed.30 IL-8 production by melanoma cells directly correlated with their metastatic potential in nude mice, as well as in humans.31,32 The production of angiogenic molecules, such as VEGF, bFGF and IL-8, by melanoma cells is regulated by complex interactions with skin keratinocytes.33 IL-8 expression, in fact, can be increased by coculturing melanoma cells with keratinocytes and inhibited by coculturing them with hepatocytes.34

VEGF in Human Melanoma

Low amounts of VEGF mRNA have been detected in normal skin.35 Salven et al demonstrated an accumulation of VEGF on the vascular endothelium in the histologically normal dermis and suggested that a constitutive low level of VEGF expression may regulate skin vessel function under normal physiological conditions.36 The expression of VEGF in melanoma has been investigated in melanoma cell lines, xenografts and primary melanoma.37-40 Salven et al reported increased VEGF expression in metastatic tumors.36 Moreover, they demonstrated that up-regulation of VEGF expression in metastatic melanoma cells was associated with an increase in the number of tumor-infiltrating inflammatory cells expressing VEGF and suggested that these cells are involved in angiogenesis. Toth et al have since used double immunolabelling to show that intradermal mast cells are the major source of VEGF around cutaneous melanoma, while their presence is also correlated with the local capillary density.41 These results illustrated the key role played by mast cells in tumor angiogenesis, by means of several angiogenic factors, such as VEGF, bFGF and tryptase, stored in their secretory granules.42 Valykova et al demonstrated by means of immunohistochemistry a high overall expression of VEGF in metastatic melanoma. The degree of VEGF positivity of the melanoma samples had a strong association with their blood vessel density.43 Straume and Akslen found that practically all vertical growth phase melanomas express VEGF to some extent, though level of expression was significantly, but inversely, related to tumor thickness and microvascular density.44 This finding suggests that VEGF may be upregulated in some smaller tumors, where lower baseline levels in thicker, more vascularized tumors may be sufficient to maintain an established vascular system by acting as a survival factor for newly-formed endothelial cells. VEGFR-1 (Flt-1) and VEGFR-2 mRNAs are strongly expressed by vascular endothelium in the numerous vessels elicited by VEGF-overexpressing melanoma cells, whereas neither is expressed in tumors formed from nontransfected cells.38 Several laboratories have identified the critical role of two other members of the VEGF cytokine family, namely VEGF-C and VEGF-D, in lymphangiogenesis.45 Mutation or loss of these cytokines or their receptor (VEGFR-3/Flt-4) leads to impaired lymphatic development, whereas their overexpression increases lymphangiogenesis in animal systems. VEGF-D is present in neoplastic, but not in normal melanocytes.46 These results suggest that it may stimulate the growth of VEGFR-3-positive lymphatic vessels in the vicinity of malignant melanoma.47 We have demonstrated a correlation between vascularization during melanoma progression and melanotransferrin and VEGF expression by their immunolocalization in sections of melanoma at different progression stages.48 We have also demonstrated that melanotransferrin is angiogenic in vitro and in vivo and that this activity is inhibited by treatment with anti-VEGF antibodies and associated with an overexpression of VEGFR-2 in newly-formed blood vessels.48 Melanotransferrin may thus be supposed to contribute to angiogenesis during melanoma progression and is probably associated with VEGF overexpression.

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Concluding Remarks The degree of in vivo angiogenesis depends on the concerted action of several pro-angiogenic and anti-angiogenic molecules. Ugurel et al have demonstrated that increased serum levels of bFGF, VEGF and IL-8 in melanoma patients are strongly correlated with poor clinical outcome and can be used to predict progression and prognosis.24 Angiogenesis in poorly angiogenic melanomas is driven by VEGF alone, whereas many factors are involved in highly angiogenic melanomas.49 The importance of angiogenesis in melanoma development is unquestionable, as well as the central role of VEGF. Consequently, the use of an anti-VEGF antibody and small molecule tyrosine kinase inhibitors to interfere with its signaling will illustrate the potential of anti-angiogenic approaches in the treatment of melanoma. Blocking VEGFR-2-mediated angiogenesis by systemic administration of a specific monoclonal antibody to animals carrying transplants of malignant keratinocytes both inhibited angiogenesis and completely abrogated tumor invasion.50 Transplants treated with control antibodies grow to form heavily vascularized invasive squamous cell carcinomas with mainly proliferating tumor and stromal cells, while this phenotype was altered in the treated animals: new blood vessel formation and penetration in the tumor stroma was abrogated.

Acknowledgements This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan), Fondazione Italiana per la Lotta al Neuroblastoma (Genoa) and Ministero dell’Istruzione, Università e Ricerca (MIUR, Rome), Italy.

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42. Ribatti D, Vacca A, Nico B et al. The role of mast cells in tumor angiogenesis. Br J Haematol 2001; 115:514-521. 43. Vlaykova T, Laurila P, Muhonen T et al. Prognostic value of tumor vascularity in metastatic melanoma and association of blood vessel density with vascular endothelial growth factor expression. Melanoma Res 1999; 9:59-68. 44. Straume O, Akslen LA. Expression of vascular endothelial growth factor, its receptors (Flt-1, KDR) and TSP-1 related to microvessel density and patient outcome in vertical growth phase melanomas. Am J Pathol 2001; 159:223-235. 45. Baldwin ME, Stacker SA, Achen MG. Molecular control of lymphangiogenesis. BioEssays 2002; 24:1030-1040. 46. Achen MG, Williams RA, Minekus MP et al. Localization of vascular endothelial growth factor-D in malignant melanoma suggest a role in tumor angiogenesis. J Pathol 2001; 193:147-154. 47. Lymboussaki A, Partanen TA, Olofsson B et al. Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors. Am J Pathol 1998; 153:395-403. 48. Sala R, Jefferies WA, Walker B et al. The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis in vivo. Eur J Cell Biol 2002; 81:599-607. 49. Rofstad EK, Halsor EF. Vascular endothelial growth factor, interleukin-8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in human melanoma xenografts. Cancer Res 2000; 60:4932-4938. 50. Skobe M, Rockwell P, Goldstein N et al. Halting angiogenesis suppresses carcinoma cell invasion. Nature Med 1997; 11:1222-1227.

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CHAPTER 7

VEGF in Esophageal Cancer Axel Kleespies, Markus Guba, Karl-Walter Jauch and Christiane J. Bruns

Abstract

E

sophageal carcinoma growth is relatively fast and patients generally have a poor prognosis. The influence of angiogenesis and pro-angiogenic molecules such as vascular endothelial growth factor (VEGF) on progression and recurrence of esophageal carcinoma has been debated over the last years. In this chapter we review published work about VEGF expression in esophageal carcinoma. VEGF contributes to the aggressive characteristics of esophageal squamous cell carcinoma (SCC) and appears to correlate with tumor stage as well as patient prognosis. Raised levels of circulating serum VEGF have been found in many esophageal carcinoma patients. In Barrett´s dysplasia and adenocarcinoma (AC) of the esophagus, angiogenic properties are acquired in the early stages of the disease, particularly in pre-cancerous lesions. However VEGF expression patterns in AC fail to give prognostic information. VEGF-C expression appears to be associated with advanced disease and lymphatic tumor invasion in esophageal SCC and AC. It remains controversial how treatment response to chemo-radiotherapy (CRT) is influenced by VEGF expression, vascular permeability or interstitial fluid pressure. Early results on experimental blockade of VEGF and its receptors are promising and gene therapy may prove to be a useful way of delivering anti-VEGF treatment.

VEGF in Squamous Cell Carcinoma of the Esophagus Esophageal carcinoma is one of the most common malignancies in the world. Esophageal carcinoma grows relatively fast, and patients generally have a worse prognosis than those with other types of gastrointestinal tumors.1 In esophageal cancer, penetration of the muscularis mucosa contributes to an increase in lymphatic metastases. This phenomenon is uncommon in other malignancies of the gastrointestinal tract, such as gastric and colonic cancer, in which lymphatic metastases are rare when the tumor is still confined to the submuscularis layer. The current staging procedure is based on the TNM classification2 and the pathological pN (node) status is currently the most powerful predictor of outcome in squamous cell carcinoma (SCC) of the esophagus.3,4 Five-year survival rates of 60-80% have been reported for pN0 (node negative) groups. Nevertheless, there are some patients with early stage esophageal cancer who die soon after curative resection because of local and distant tumor recurrence. Recurrence rates of 8%-20% are reported for pT1 esophageal SCC.5 The influence of angiogenesis and pro-angiogenic molecules on progression and recurrence of esophageal carcinoma has been debated over the last few years. While only a few studies have been published regarding vascular endothelial growth factor (VEGF) expression in Barrett’s adenocarcinoma (AC), an increasing number of articles deal with VEGF expression and microvessel assessment in squamous cell carcinoma of the esophagus (SCC). In this chapter we review published work concerning VEGF in SCC. All recent studies which addressed expression patterns of vascular endothelial growth factor in esophageal SCC, revealed VEGF-immunoreactivity to some degree.6-22 The proportion of VEGF VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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positive staining tumors varies between 31% to 65% in those studies.7,9 Biopsies taken either pre-operatively or prior to chemo-radiation therapy yielded similar results. 19,22 Correspondingly, VEGF gene expression in esophageal SCC could be demonstrated using reverse transcriptase polymerase chain reaction (RT-PCR) for VEGF mRNA.18 Other authors discovered elevated VEGF levels in serum samples of patients suffering from esophageal SCC.13,20,21 Several authors additionally assessed vascularization of esophageal squamous cell carcinoma. The majority—eight out of nine—could show a certain association between VEGF protein expression and microvessel density (MVD) in SCC.7,10,12,17,18,22 However, this correlation was only statistically significant in four publications (33%).10,16,18,22 Prognostic significance of MVD measurement was established in three populations of SCC patients,9,16,19 while two papers found no predictive value of MVD in esophageal carcinoma.12,22 Nonetheless, in about 83% of cases VEGF immunoreactivity was strongly associated with tumor stage, spread or progression.7-10,13,14,16-18,20 Eight out of twelve authors (66%) describe a positive correlation between VEGF expression and presence of local lymph node metastases.7-10,13,14,17,20 Similarly, a significant association between VEGF expression and pT score or presence of distant metastases (M+ stage) was demonstrated in five and four studies, respectively. Only three authors evaluated vascular infiltration, and all of them found a positive relationship between angioinvasion and VEGF protein expression in esophageal SCC. One author also found a positive correlation between VEGF and pathological grade of malignancy.18 Only two of twelve papers (16%) found no association between VEGF expression and tumor stage or grade of malignancy.12,21 If one considers that positive lymph node status is a proven predictor of poor outcome in esophageal carcinoma, and that high VEGF expression seems to be associated with positive lymph nodes, then VEGF measurement should be a prognostic tool to predict long term survival. In support of this ten out of thirteen studies (78%) found that VEGF protein expression correlated with patient outcome.6,7,9,10,15-18,20,22 In various studies VEGF was a predictor of poor survival in univariate analysis, however a number of studies describe VEGF measurement in esophageal SCC as an independent prognostic parameter when evaluated by multivariate analysis.9,10,15 VEGF is a multifactorial growth factor which promotes stromal degeneration through activation of proteolytic enzymes,23 causes endothelial sprouting through induction of endothelial cell migration and proliferation,24 and facilitates extravasation of molecules and cells via enhancement of vascular permeability.25 Therefore, VEGF strongly contributes to angiogenesis and presumably to tumor growth and hematogenous tumor spread. However, to date it is not clear which of VEGF´s functions plays the most important role in the development of local lymph node metastases, which are significant predictors of poor prognosis in esophageal SCC and are associated with increased VEGF expression. Early tumor spread to local lymph nodes could be interpreted as a secondary effect of accelerated tumor growth and invasiveness induced primarily by angiogenesis due to VEGF over-expression. If this is so then VEGF leads to early metastasis, when it is abundant in an early stage of esophageal SCC, while late stage metastases seem to be VEGF independent.14 However, most of the above mentioned studies confirm that VEGF contributes to the aggressive characteristics of esophageal squamous cell carcinoma and appears to correlate with tumor stage. While the predictive value of VEGF measurement has been argued over the last years, VEGF expression appears to correlate with patient outcome at this time.

VEGF in Barrett’s Disease and Adenocarcinoma of the Esophagus Barrett’s metaplasia of the lower esophagus is one of the most common pre-cancerous lesions in the western world and its incidence is increasing. A clonal evolution of mucin secreting cells within the native squamous mucosa arises as a consequence of gastro-esophageal reflux disease (GERD). Once GERD has led to the onset of metaplastic mucosa, metaplasia appears to never totally regress.26 Harmful bile and acid reflux alters not only the epithelium but also the lamina propria. In addition to epithelial cells, Barrett’s metaplasia is also composed of

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inflammatory cells and endothelial cells of the lamina propria. Metaplastic cells were shown to secrete VEGF,27,28 perhaps as a consequence of bidirectional secretion of tumor necrosis factor alpha (TNFα) and transforming growth factor alpha (TGFα) by inflammatory cells and damaged epithelial cells, respectively.26 However, little is known about the onset and role of pathological angiogenesis in early and advanced Barrett's carcinoma. In animal experiments Baatar et al induced esophageal ulcers in rats by focal acid application. Those ulcerations caused hypoxia-inducible factor 1α (HIF-1α) protein expression in microvessels and subsequent VEGF gene activation in cells bordering necrotic areas, reflected by increasing VEGF mRNA and VEGF protein levels. This reaction indicates a role for VEGF expression in esophageal ulcer healing. Auvinen et al28 studied surgically resected human Barrett’s dysplasia and saw the characteristic flames of “salmon-colored” mucosa, thought to be a consequence of continuous erosion as a consequence of chronic reflux. However, immunohistochemistry revealed that the “salmon-red” color of the mucosa was due to incipient angiogenesis, which infiltrates Barrett’s epithelium and originates from the pre-existing vascular network in the lamina propria. Barrett’s epithelial cells expressed VEGF, while VEGF receptor 2 (VEGFR-2) and metalloproteinase 9 (MMP-9) were found on immature blood vessels. Therefore, Barrett’s esophagus is strongly neovascularized and not simply eroded. In a study of seventy-eight patients with Barrett’s adenocarcinoma VEGF protein expression was apparent in metaplastic and neoplastic epithelium when studied immunohistochemically.27 Moreover, VEGF expression correlated significantly with microvascularization in both metaplasia and tumor. The authors report stepwise increasing microvessel counts in high-grade dysplastic mucosa, intra-mucosal carcinoma and superficial carcinoma (pT1), respectively, when compared with non-dysplastic Barrett’s mucosa. In contrast, microvessel counts were reduced in infiltrative carcinoma. Paradoxically, high vascularization was associated with a lower rate of lymph node and distant metastases as well as with better survival. However, this observation was due to the correlation of high vascularization and superficial cancers, as mentioned above. When early carcinomas were excluded from the study, VEGF and microvessel count failed to provide any prognostic information in invasive cancer.27 This result corresponds to findings of Torres et al, who did not identify a prognostic value for microvessel density in 45 Barrett’s carcinomas.30 In conclusion, VEGF expression in Barrett’s dysplasia and Barrett’s carcinoma seems to correlate with vascularization. Angiogenic properties are acquired early, particularly in pre-cancerous lesions and in superficial cancers, representing a critical step in the development of Barrett’s adenocarcinoma. Angiogenesis might occur as early as the transition from metaplasia to neoplasia.31 This finding could provide one possible explanation for the early onset of local spread and frequent recurrence of esophageal carcinoma. However, VEGF expression patterns and microvessel density apparently fail to give prognostic information in invasive adenocarcinoma of the esophagus.

Circulating VEGF Levels in Esophageal Carcinoma Angiogenic activity of malignant tumors can be estimated by immunohistochemical evaluation of microvessel density, assessment of paracrine growth factors such as VEGF, basic fibroblast growth factor (bFGF) or TGFα and their respective receptors or by measurement of gene expression via in situ-hybridization and PCR. All these procedures require a tissue specimen of the tumor of interest. However, elevated levels of pro- and anti-angiogenic factors have also been detected in biological fluids such as blood, urine, cerebro-spinal fluid or pleural and peritoneal effusions of tumor patients.32-39 It remains unclear whether this phenomenon represents an active endocrine expression of molecules, or just a pathological passive release of those molecules by apoptotic tumor cells or hematological effector cells such as macrophages and platelets32,40,41 during inflammatory reactions. Raised levels of circulating VEGF measured with enzyme-linked immunosorbent assay kits (ELISA) were reported in various types of cancer42-44 and were also associated with a short tumor-volume doubling time45 and poor outcome.39,42,46-49 Only a small number of articles describe circulating VEGF levels in esophageal carcinoma.15,20,21,50

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Shimada et al20 analyzed serum VEGF (S-VEGF) concentration in 82 patients with primary SCC of the esophagus, 14 patients with recurrent esophageal carcinoma and 24 healthy controls. S-VEGF was found to be significantly elevated in patients with primary SCC but not in recurrent disease or healthy controls. Moreover S-VEGF correlated significantly with tumor size, tumor stage, nodal status and distant metastases status but not with gender or age. High serum levels of VEGF were associated with poor survival. Furthermore, a multivariate analysis with respect to survival rate revealed S-VEGF to be slightly less significant than tumor stage in this study.20 McDonnell et al21 also detected elevated S-VEGF levels in both squamous cell (n=20) and adenocarcinoma (n=24) of the esophagus compared to healthy controls (n=20). However, they did not find a correlation between S-VEGF levels and tumor stage or survival. S-VEGF levels were unaffected by peri-operative chemo-radiotherapy (CRT); values remained high throughout the whole course of neo-adjuvant treatment but were reduced after surgery. Therefore the authors speculate that VEGF identified in serum of these patients not only represents VEGF secreted by tumor cells, but also VEGF secreted by tumor-associated macrophages. The number of macrophages was largely unaffected by neo-adjuvant treatment, but they were removed by surgery. The stimulatory effect of tumor-associated macrophages on tumor angiogenesis has recently been identified;51 macrophages are potent producers of VEGF.36,52,53 Their VEGF production is up-regulated by tumor hypoxia and transforming growth factor β1 (TGFβ1), both of which are present in the central tumor environment. On the other hand tumor cell apoptosis or hypoxic cell lysis could result in elevated VEGF as a result of passive VEGF release.54 Spence et al implicated ex vivo activation of platelets in serum samples as an artificial source of elevated VEGF levels in EC patients due to their passive VEGF release. Alterations in S-VEGF levels might, therefore, be influenced more by changes in platelet number than by tumor effects; and measurement of plasma-VEGF (P-VEGF) instead of serum-VEGF (S-VEGF) should be more specific. Nevertheless, platelets are also activated in the tumor vasculature of cancer patients in vivo and will consequently release VEGF in vivo.55 Moreover pre- and postoperative P-VEGF levels were also found to correlate with platelet counts in 23 patients undergoing esophageal resection.56 Therefore platelets may contribute to VEGF levels in plasma as well as in serum. Regardless of the source (macrophages, growing tumor cells, apoptotic tumor cells or platelets), raised levels of circulating VEGF could be found in most esophageal cancer patients. Moreover, high circulating VEGF levels may also be responsible for the growth of micrometastases at distant sites.57 This hypothesis has clinical implications because micrometastases have been identified in the bone marrow of more then 80% of patients suffering from gastro-esophageal carcinoma.58

VEGF and MVD during Neoadjuvant Treatment of Esophageal Carcinoma Esophageal carcinoma is still one of the most lethal malignancies among gastrointestinal carcinomas. Surgery is performed as standard treatment but prognosis for patients remains poor with 5 year survival rates of only 10-20%. Different multimodality approaches have been investigated to improve outcome of patients and various reports on pre-operative chemo-radiotherapy have indicated advantages for managing esophageal carcinoma.59-62 A combination therapy of 5-Fluorouracil (5-FU) and Cisplatin exerts anti-neoplastic effects and enhances radio-sensitivity. It remains unclear whether definitive CRT without subsequent surgery is potentially curative.63 However, improved survival has consistently been reported in patients in whom pre-operative CRT led to a complete tumor response, defined as the absence of vital tumor cells within the resected specimen.64,65 For those patients, 3 year survival rates of more than 50% can be expected. Complete response is reported in 20-30% of cases after neo-adjuvant CRT, regardless of the applied protocol, histology (SCC vs. AC) or tumor stage. On the other hand peri-operative morbidity and mortality are increased by CRT. This side effect is relevant for all patients including those who show only partial or no tumor response to

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CRT. Therefore identification of factors which could predict a response to CRT is required. Some markers including p53, Ki-67 (a marker of proliferating cells) and epidermal growth factor receptor (EGF-R) seem to have prognostic value in patients receiving CRT for esophageal carcinoma.66-68 Tissue oxygen status has been demonstrated to be a very important factor in determining radiosensitivity both in vitro and in vivo.69,70 Only a well oxygenated cell is fully radiosensitive.71 On the other hand tumor microcirculation and vessel permeability are important factors for delivery of drugs and radiosensitizers to cancer cells. Thereby the prognostic value of MVD assessment for CRT response could be explained. VEGF expression has been shown to induce angiogenesis and vascular permeability. Consequently the level of VEGF expression in esophageal cancer may be of value in predicting response to CRT. However, VEGF expression and subsequent microvessel sprouting are also responsible for tumor nutrition, growth, local invasion and metastatic spread. Therefore, conflicting results for the prognostic value of VEGF and MVD during CRT could be expected. In a retrospective study, Hironaka et al analyzed immunohistochemical markers in pretreatment biopsy specimens from 73 SCC patients before definitive CRT (5-FU, Cisplatin, 60 Gy).19 Response to CRT was assessed clinically by time of survival. Although this study identified a weak association between p53 and Ki-67 and survival, only MVD and pT stage were independent prognostic variables for survival time using multivariate analysis. VEGF expression, measured semi-quantitatively as VEGF-positive staining of tumor cytoplasm, was reported in 49 percent of the patients but did not show a correlation with survival time or with any other clinicopathological marker. In contrast to former studies 9.30,72 patients with high MVD tumors survived significantly longer than those with low MVD tumors. This observation appears plausible since Hironka et al assessed MVD by counting microvessels with visible lumens only.19 The authors conclude that lumen-MVD should be suited to select patients for CRT because lumen-MVD rather than total MVD is a marker of the oxygenation status of the tumor. On the other hand we know that high vascular density is not synonymous with high blood flow, tissue oxygenation or drug delivery. A non-functional geometry of the abnormal tumor vasculature and an immature structure of the vessels may result in an impaired blood flow.73 Moreover uncontrolled angiogenesis leads to elevated interstitial fluid pressure (IFP) as a result of an increasing vascular surface and vascular permeability without an adequate lymphatic drainage of the tumor.74 Together these factors may result in reduced drug delivery to the tumor, decreased tissue oxygenation and resistance to CRT. In contrast the results of Imdahl et al22 suggest that tumors with low MVD respond better to CRT than those with high vessel density. Weak VEGF immunoreactivity in pre-treatment biopsies was associated with complete tumor response after neoadjuvant CRT (5-FU, Cisplatin, 36 Gy) in 56 patients with SCC or adenocarcinoma of the esophagus. Moreover, reduced VEGF expression led to better long term survival after CRT and surgery. However, no correlation between clinical staging and CRT response could be established. MVD showed a weak correlation with VEGF expression and tumor response but not with long term survival. A high proliferation index, measured as Ki-67 positive staining of tumor cells, was associated with a complete response and was an independent factor for survival in multivariate analysis, suggesting that tumors with an increased cell turnover respond more rapidly to CRT. Comparable results were found in two recent studies by Shimada et al,20,75 where high amounts of pretreatment serum-VEGF were associated with tumor progression, poor response to CRT (5-FU, Cisplatin, 40 Gy) and poor survival in 35 patients with SCC of the esophagus. Response to CRT treatment was assessed clinically by image analysis and endoscopic reassessment. Pretreatment S-VEGF levels and tissue-VEGF expression (measured immunohistochemically after surgery) were significantly higher in non-responders than in individuals responding to CRT. The 3 year and 5 year survival rate was significantly lower in patients with high serum VEGF levels or VEGF positive staining carcinomas, respectively. No alteration of serum-VEGF levels by neo-adjuvant CRT treatment (5-FU, Cisplatin, 40 Gy) was detected in the study by McDonnell et al.21 These authors quantified S-VEGF levels at various days before, during and

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after CRT and surgery in 20 patients with SCC, and 24 patients with adenocarcinoma of the esophagus. S-VEGF was elevated in tumor patients versus healthy controls and S-VEGF levels decreased one day after surgery reflecting the reduction of tumor mass. Elevation of S-VEGF was seen during wound healing from day 3 on and S-VEGF decreased below pretreatment levels three months after surgery. Nevertheless no correlation between S-VEGF levels during CRT and tumor response to CRT was found. Thus, the authors conclude that there must be an additional source of S-VEGF which is not affected by CRT in those patients (e.g., macrophages). In conclusion, VEGF expression and vascularization seem to play a role in growth and spread of esophageal carcinoma, as well as in drug delivery to, and oxygenation of, the tumor cells. How treatment response to chemo-radiotherapy is influenced by VEGF expression, vessel density, vascular permeability and interstitial fluid pressure remains controversial.

VEGF-C and Lymphangiogenesis in Esophageal Carcinoma A special feature of esophageal carcinoma is its early lymphatic spread into local lymph nodes when compared with other malignancies of the upper gastrointestinal tract. Lymph vessel invasion of cancer cells has been noticed for a long time, and can be assessed by light-microscopy. However, lymphangiogenesis as an active process, driven by specific growth factors under specific conditions during neoplastic transformation was only recently established.76-78 VEGF-C and VEGF-D, both members of the VEGF family, bind specifically to VEGF-R3 (Flt-4), another tyrosine kinase receptor which is predominantly expressed on lymphatic endothelium.79-81 Activation of VEGFR-3 by ligand binding induces lymphatic vessel growth or so-called lymphangiogenesis, but has very little effect on blood capillaries. To date no studies are published about VEGF-D expression and lymph node metastases in esophageal carcinoma. Nonetheless, the following studies suggest that VEGF-C plays a role in lymphatic invasion, lymphangiogenesis and early lymph node metastases in esophageal carcinoma. Noguchi et al evaluated expression of VEGF-C and its receptor Flt-4 (VEGFR-3) in esophageal carcinoma cell lines, pre-operative biopsies and surgical specimens of resected SCC of the esophagus.82 Four out of five cell lines expressed VEGF-C mRNA (80%) and 8 out of 12 biopsies revealed VEGF-C expression at the mRNA level (67%) by RT-PCR. Normal and dysplastic mucosa did not exhibit VEGF-C protein or gene expression, but 29 out of 48 surgical carcinoma specimens (60 %) revealed VEGF-C protein expression in cancer cell cytoplasm or stromal cells, visualized by immunohistochemistry. In contrast Flt-4 (VEGFR-3) was mainly expressed on lymphatic endothelium as the target cell compartment. The authors found significant positive correlations between VEGF-C expression and tumor stage, depth of the tumor, vascular- and lymphatic invasion and lymph node metastases.82 Moreover microvessel count was higher in VEGF-C positive tumors. This finding may result from VEGF-C binding to VEGFR-2 (KDR), which is mainly expressed on endothelial cells in blood vessels. In contrast, Kitadai et al contest the prognostic value of VEGF-C expression in esophageal SCC.83 In their recent paper the authors could not find any significant correlation between VEGF-C expression as assessed by immunohistochemistry and any clinicopathological parameter except tumor grade in seventy-one SCC patients. Only histological differentiation (grading) was loosely associated with VEGF-C immunoreactivity. Auvinen et al28 identified stepwise increasing VEGF-C expression during progression from Barrett’s epithelium to dysplasia and to Barrett’s adenocarcinoma. No VEGF-C expression was found in normal esophageal mucosa. VEGFR-3 expression on lymphatic vessels was up-regulated during development of esophageal adenocarcinoma. Additionally, lymphatic vessels were found to penetrate tumor stroma and VEGF-C and VEGFR-3 expression was identified in metastatic lymph nodes. VEGF-C appears to be expressed in esophageal squamous cell and adenocarcinoma by cancer cells and possibly also by stromal cells. Expression of VEGF-C and its receptor Flt-4 on lymphatic endothelium is probably associated with advanced disease in human esophageal carcinoma and may be involved in lymphatic tumor invasion and lymphangiogenesis in esophageal carcinoma. This mechanism might provide another explanation for the early

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onset of lymphatic spread in some esophageal carcinomas, a phenomenon which predicts a poor outcome.

Anti-VEGF Treatment of Esophageal Carcinoma Since VEGF is a pro-angiogenic molecule which may facilitate tumor growth and spread, blocking its angiogenic effects might be a promising tool to slow down or even stop tumor progress. Several studies were undertaken to show efficacy of anti-VEGF treatment in tumor models. In general, these studies tested neutralizing antibodies against mature VEGF protein or one of its isoforms, blockade of VEGF receptors by VEGF receptor antibodies, soluble VEGF receptor mutants or fusion-proteins and finally, intracellular interference with VEGF mRNA or signal transduction in the target cell. Until now only a few authors studied options to interfere with VEGF expression in esophageal carcinoma. Regarding acid-induced esophageal ulcers, Baatar et al29 enhanced angiogenesis and accelerated ulcer healing in rats by local injection of plasmid cDNA encoding recombinant human VEGF165 isoform. In contrast Gu et al reduced VEGF165 expression in an esophageal squamous cell carcinoma cell line EC109 by transfection of VEGF165 antisense-RNA.84 VEGF165 expression was significantly decreased while the biologic characteristics of the cells were largely unaffected. When transplanted into nude mice, the tumorigenic and angiogenic capability of the tumor were significantly reduced, as demonstrated by smaller tumor volume and decreased microvessel density.84 Another group demonstrated that the anti-tumor effects of VEGF165 antisense could be improved by placing the antisense construct under the control of a hypoxia response element (HRE).85 This effect occurred because the HRE drives expression of VEGF165 antisense construct in hypoxic areas of the tumor where VEGF expression is maximal. Taken together, early results on the anti-tumor efficacy of VEGF blockade or blockade of VEGF binding to its receptors in esophageal cancer cells and/or experimental tumor models are promising. A reduction in tumor angiogenesis and tumor growth may be anticipated and gene therapy may yet prove to be a promising way of delivering anti-VEGF treatment. However, concrete clinical data are still pending.

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13. Wallner G, Ciechanski A, Dabrowski A et al. Vascular endothelial growth factor and basic fibroblast growth factor in patients with squamous cell oesophageal cancer. Folia Histochem Cytobiol 2001; 39 Suppl2:122-123. 14. Sato F, Shimada Y, Watanabe G et al. Expression of vascular endothelial growth factor, matrix metalloproteinase-9 and E-cadherin in the process of lymph node metastasis in oesophageal cancer. Br J Cancer 1999; 80:1366-1372. 15. Shimada Y, Imamura M, Watanabe G et al. Prognostic factors of oesophageal squamous cell carcinoma from the perspective of molecular biology. Br J Cancer 1999; 80:1281-1288. 16. Kitadai Y, Haruma K, Tokutomi T et al. Significance of vessel count and vascular endothelial growth factor in human esophageal carcinomas. Clin Cancer Res 1998; 4:2195-2200 17. Uchida S, Shimada Y, Watanabe G et al. In esophageal squamous cell carcinoma vascular endothelial growth factor is associated with p53 mutation, advanced stage and poor prognosis. Br J Cancer 1998; 77:1704-1709. 18. Inoue K, Ozeki Y, Suganuma T et al. Vascular endothelial growth factor expression in primary esophageal squamous cell carcinoma. Association with angiogenesis in tumor progression. Cancer 1997; 79:206-213. 19. Hironka S, Hasebe T, Kamijo T et al. Biopsy specimen microvessel density is a useful prognostic marker in patients with T(2-4) M(0) esophageal cancer treated with chemoradiotherapy. Clin Cancer Res 2002; 8:124-130. 20. Shimada H, Takeda A, Nabeya Y et al. Clinical significance of serum vascular endothelial growth factor in esophageal squamous cell carcinoma. Cancer 2001; 92:663-669. 21. McDonnell Co, Harmey JH, Bouchier-Hayes DJ et al. Effect of multimodality therapy on circulating vascular endothelial growth factor levels in patients with esophageal cancer. Br J Surg 2001; 88:1105-1109. 22. Imdahl A, Bognar G, Sculte-Monting J et al. Predictive factors for response to neoadjuvant therapy in patients with oesophageal cancer. Eur J Cardiothorac Surg 2002 ; 21 :657-663. 23. Ferrara N. Vascular endothelial growth factor. Eur J Cancer 1996:32a:2413-2422. 24. Senger DR, Van de Water L, Brown LF et al. Vascular permeability factor (VPF, VEGF) in tumor biology. Cancer Metastasis Rev 1993; 12:303-324. 25. Roberts WG and Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci 1995; 108:2369-2379. 26. Harrison RF, Perry I, Jankowski JA et al. Barrett’s mucosa: remodeling by the microenvironment. J Pathol 2000; 192:1-3. 27. Couvelard A, Paraf F, Gratio V et al. Angiogenesis in the neoplastic sequence of Barrett’s oesophagus. Correlation with VEGF expression. J Pathol 2000; 192:14-18. 28. Auvinen MI, Sihvo EI, Ruohtula T et al. Incipient angiogenesis in Barrett‘s epithelium and lymphangiogenesis in Barrett’s adenocarcinoma. J Clin Oncol 2002; 20:2971-2979. 29. Baatar D, Jones MK, Tsugawa K, et al. Esophageal ulceration triggers expression of hypoxia-inducible factor-1 alpha and activates vascular endothelial growth factor gene :implications for angiogenesis and ulcer healing. Am J Pathol. 2002; 161:1101-1105. 30. Torres C, Wang H, Turner J et al. Prognostic significance and effect of chemoradiotherapy on microvessel density (angiogenesis) in esophageal Barrett’s esophagus associated adenocarcinoma and squamous cell carcinoma. Hum Pathol 1999; 30:753-758. 31. Folkman J, Watson K, Ingber D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989; 339:58-61. 32. Dunque JL, Loughlin KR, Adam RM et al. Plasma levels of vascular endothelial growth factor are increased in patients with metastatic prostate cancer. Urology 1999; 54:523-527. 33. Weingärtner K, Ben-Sasson SA, Stewart R et al. Endothelial cell proliferation activity in benign prostatic hyperplasia and prostate cancer: an in vitro model for assessment. J Urol 1998; 159:465-470. 34. Nguyen M, Watanabe H, Budson AE et al. Elevated levels of angiogenic peptide, basic fibroblast growth factor, in urine of patients with a wide spectrum of cancers. J Natl Cancer Inst; 86:356-361. 35. Li VW, Folkert RD, Watanabe H. Microvessel count and cerebrospinal fluid basic fibroblast growth factor in children with brain tumors. Lancet 1994; 344:82-86. 36. Yeo KT, Wang HH, Nagy JA. Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions. Cancer Res 1993; 53:2912-2918. 37. O´Reilly MS, Holmgren L, Shing Y et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79:315-328. 38. Holmgren L, O´Reilly MS, Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1995; 1:149-153.

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39. Kraft A, Weindel K Ochs A et al. Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease. Cancer 1999; 85:178-187. 40. Mohle R, Green D, Moore MA et al. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci USA 1997; 94:663-668. 41. Banks RE, Forbes MA, Kinsey SE et al. Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer 1998; 77:956-964. 42. Yamamoto Y, Toi M, Kondo S et al. Concentrations of vascular endothelial growth factor in the sera of normal controls and cancer patients. Clin Cancer Res 1996; 2:821-826. 43. Kondo S, Asano M, Matsuo K et al. Vascular endothelial growth factor/ vascular permeability factor is detectable in the sera of tumor bearing mice and cancer patients. Biochem Biophys Acta 1994; 1221:211-214. 44. Fujisaki K, Mitsuyama K, Toyonaga A et al. Circulating vascular endothelial growth factor in patients with colorectal cancer. Am J Gastroenterol 1998; 93:249-252. 45. Dirix LY, Vermeulen PB, Hubens G et al. Serum basic fibroblast growth factor and vascular endothelial growth factor and tumor growth kinetics in advanced colorectal cancer. Ann Oncol 1996; 7:843-848. 46. Salven P, Routsalainen T, Mattson K et al. High pretreatment serum vascular endothelial growth factor (VEGF) is associated with poor outcome in small-cell lung cancer. Int J Cancer 1998; 79:144-146. 47. Salven P, Teerenhovi L, Joensuu H et al. High pretreatment serum vascular endothelial growth factor concentration is associated with poor outcome in non-Hodgkin’s lymphoma. Blood 1997; 90:3167-3172 48 Kumar H, Heer K, Lee PWR et al. Preoperative serum vascular endothelial growth factor can predict stage in colorectal cancer. Clin Cancer Res 1998; 4:1279-1285. 49 Hefler L, Tempfer C, Obermair A et al. Serum concentration of vascular endothelial growth factor in vulvar cancer. Clin Cancer Res 1999; 5:2806-2809. 50 Linder C, Linder S, Muck Wikeland E et al. Independent expression of serum vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in patients with carcinoma and sarcoma. Anticancer Res 1998; 18:2063-2068. 51 Lewis CF, Leek R, Harris A et al.Cytokine regulation of angiogenesis in breast cancer: the role of tumor associated macrophages. J Leukoc Biol 1995; 57:747-751. 52. Berse B, Brown LF, Van de Water L et al.Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages and tumors. Mol Biol Cell 1992; 3:211-220. 53. Sheid B. Angiogenic effects of macrophages isolated from ascitic fluid aspirated from women with advanced ovarian cancer. Cancer Lett 1992; 62:153-158. 54. Spence GM, McAllister I, Graham AN et al. Comment on: “Effect of multimodality therapy on circulating vascular endothelial growth factor levels in patients with esophageal cancer”. Br J Surg 2002; 89:495-496. 55. Verhaul HM, Hoekman K, Lupu F et al. Platelet and coagulation activation with vascular endothelial growth factor generation in soft tissue sarcomas. Clin Cancer Res 2000; 6:166-171. 56. Spence GM, Graham AN, Mulholland K et al. Vascular endothelial growth factor levels in serum and plasma following esophageal cancer resection—Relation to platelet count. In J Biol Markers 2002; 17:119-124. 57. Maniwa Y, Okada M, Ishii N et al. Vascular endothelial growth factor increased by pulmonary surgery accelerates the growth of micrometastases in metastatic lung cancer. Chest 1998; 114:1668-1675. 58. O´Sullivan GC, Sheehan D, Clarke A et al. Micrometastases in esophagogastric cancer: high detection rate in resected rib segments. Gastroenterology 1999; 116:543-548. 59. Vogel SB, Medenhall WM, Sombeck MD et al. Downstaging of esophageal cancer after preoperative radiation and chemotherapy. Ann Surg 1995; 221:685-695. 60. Ohtsu A, Yoshida A, Boku N et al. Concurrent chemotherapy and radiation therapy for locally advanced carcinoma of the esophagus. Jpn. J Clin Oncol 1995; 25:261-266. 61. Coia LR. Chemoradiation as primary management of esophageal cancer. Semin Oncol 1994; 21:483-492. 62. Walsh TN, Noonan N, Hollywood D et al. A comparison of multimodal therapy and surgery for esophageal adenocarcinoma. N Engl J Med 1996; 335:462-467. 63. Chan A and Wong A. Is combined chemotherapy and radiation therapy equally effective as surgical resection in localized esophageal carcinoma? Int J Radiat Oncol Biol Phys 1999; 45:265-270.

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64. Lackey VL, Reagan MT, Smith RA et al. Neoadjuvant therapy in squamous cell carcinoma of the esophagus: role of resection and benefits in partial responders. Ann Thorac Surg 1989; 48:218. 65. Bosset JF, Gignoux M, Triboulet JP et al. Chemoradiotherapy followed by surgery compared with surgery alone in squamous cell carcinoma of the esophagus. N Engl J Med 1997; 337:161-167. 66. Kitamura K; Saeki H, Kawaguchi H et al. Immunohistochemical status of the p53 protein and Ki67 antigen using biopsy specimens can predict a sensitivity to neoadjuvant therapy in patients with esophageal cancer. Hepatogastroenterology 2000; 47:419-423. 67. Yang B, Rice TW, Adelstein DJ et al. Overexpression of p53 protein associates decreased response to chemoradiotherapy in patients with esophageal carcinoma. Mod Pathol 1999; 12:251-256. 68. Hickey K, Grehan D, Reid IM et al. Expression of epidermal growth factor receptor and proliferating cell nuclear antigen predicts response of esophageal squamous cell carcinoma to chemoradiotherapy. Cancer 1994; 74:1693-1698. 69. Rockwell S and Moulder JE. Hypoxic fractions of human tumors xenografted into mice: a review. Int J Radiat Oncol Biol Phys 1990; 19:197-202. 70. Moulder JE and Rockwell S. Tumor hypoxia: its impact on cancer therapy. Cancer Metastasis Rev 1987; 5:331-341. 71. Suit HD and Suchato C. Hyperbaric oxygen and radiotherapy of a fibrosarcoma and of a squamous-cell carcinoma of C3H mice. Radiology 1967; 89:713-719. 72. Tanigawa N, Matsumura M, Amaya H et al. Tumor vascularity correlates with the prognosis of patients with esophageal squamous cell carcinoma. Cancer 1997; 79:220-225. 73. Koukourakis MI. Tumor angiogenesis and response to radiotherapy. Anticancer Res 2001; 21:4285-4300. 74. Boucher Y, Leunig M, Jain RK. Tumor angiogenesis and interstitial hypertension. Cancer Res 1996; 56:4264-4266. 75. Shimada H, Hoshino T, Okazumi S et al. Expression of angiogenic factors predicts response to chemoradiotherapy and prognosis of oesophageal squamous cell carcinoma. Br J Cancer 2002; 86(4):552-557 76. Kapanen T, Egeblad M, Karkkainen M et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res 2001; 61:1786-1790. 77. Oh SJ Jeltsch MM, Birkenhager R et al. VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev Biol 1997; 188:96-109. 78. Jeltsch M, Kaipainen A, Joukov V et al. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 1997; 276:1423-1425. 79. Kaipainen A Korhonen J, Mustonen T et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA 1995; 92:3566-3570. 80. Kukk E, Lymboussaki A, Taira S et al. VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 1996; 122:3892-3837. 81. Dumont DJ, Jussila L, Taipale J et al. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 1998; 282:946-949. 82. Noguchi T, Takeno S, Shibata T et al. VEGF-C expression correlates with histological differentiation and metastasis in squamous cell carcinoma of the esophagus. Oncol Rep 2002; 9(5):995-999. 83. Kitadai Y, Amikoka T, Haruma K et al. Clinicopathological significance of vascular endothelial growth factor (VEGF)–C in human esophageal squamous cell carcinomas. Int J Cancer 2001; 93:662-666. 84. Gu ZP, Wang YJ, Li JG et al. VEGF165 antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma. World J Gastroenterol 2002; 8:44-48. 85. Guo WZ, Ran YL, Liu J et al. Enhancement by hypoxia of antisense VEGF(165) gene expression in esophageal cancer cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao 2002; 34:625-629.

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CHAPTER 8

VEGF in Colorectal Cancer Markus Guba, Hendrik Seeliger, Karl-Walter Jauch and Christiane J. Bruns

Abstract

A

ngiogenesis plays an important role in colorectal cancer progression. Evidence from preclinical and clinical studies indicates that vascular endothelial growth factor (VEGF) is the predominant angiogenic factor in human colorectal cancer and is associated with formation of metastases and poor prognosis. Based on these results it was hypothesized that attacking one or more of the VEGF-mediated mechanisms may be promising in the treatment of colorectal cancer. This article reviews the role of VEGF in colon cancer and summarizes recent advances in the treatment of colorectal cancer by anti-VEGF strategies.

Colon Cancer Colorectal cancer is the second-leading cause of cancer-related deaths in Europe and the US. Worldwide, more than 900,000 new cases of colorectal cancer are diagnosed each year and colorectal cancer accounts for nearly 500,000 cancer deaths annually.1 Approximately 30% of patients diagnosed with colorectal cancer already have metastases at first presentation. Screening programs can however detect early stage colorectal carcinomas when they are most curable. Colonic polyps are the likely precursor to the development of cancer in most patients. Advances in the understanding of the molecular events involved in the development of colon carcinoma led to the development of agents that may prevent the development of colorectal cancer. Celecoxib (Celebrex) is a cyclooxygenase 2 (COX-2) inhibitor which can potentially decrease the development of pre-malignant polyps in patients with an inherited form of colon polyps, familial adenomatous polyposis (FAP), which is associated with an elevated risk of colon cancer.2 In the case of manifest colorectal cancer, currently the most important factor predicting survival is the regional lymph node status at the time of initial surgery. However, this is not sufficient to predict outcomes accurately. Approximately 20 % of patients with stage II disease (without regional lymph node involvement) and approximately 50 % of patients with Stage III disease (with positive regional lymph nodes) will not be alive 5 years after curative resection. Although much has been learned regarding the molecular pathogenesis of colorectal carcinoma in the past decades, the elucidation of prognostic markers that could also serve as therapeutic targets is necessary to better understand and improve outcomes.

VEGF in Colorectal Cancer Progression In colorectal cancer vascular endothelial growth factor (VEGF) levels are elevated and correlated with a poor clinical outcome.3 However, angiogenesis is not restricted to the advanced stages of cancer but can also be observed early in pre-malignant stages of tumor development. Recently it has been shown that VEGF expression in benign colonic adenomas is significantly upregulated compared to normal colonic mucosa, with a further increase during the development of adenocarcinomas. Furthermore, it has been shown that within the tumor VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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the tumor cells had the highest expression of VEGF, and high VEGF expression by the tumor cells is required to establish the invasive phenotype.4,5 Interestingly, VEGF protein levels and blood vessel counts are similar in adenomas and non-metastatic colorectal malignancies,3 whereas blood vessel counts and VEGF levels in metastatic colorectal tumors are significantly higher. The mechanisms that up-regulate VEGF in cancerous states are complex. Hypoxia inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of the basic helix-loop-helix proteins HIF-1α and HIF-1β, is one key regulator.6 Although mutant H-Ras regulates VEGF in epithelial, fibroblast, and endothelial cell lines,7 the relevance to gastrointestinal malignancies is unclear, because mutations in K-ras are almost exclusively identified in colon polyps and colon cancer. Inhibition of the phosphatidylinositol 3’-kinase (PI3-K) signalling pathway with wortmannin can reduce the stimulatory effect of H-ras in endothelial cells8 but independent studies have demonstrated that the ERK-1/2 (p42/p44 Mitogen Activated Protein (MAP) kinase) signalling pathway is more relevant in terms of VEGF expression in fibroblasts.9 Thus, the pathways that regulate VEGF expression are cell-specific. In colon cancer, activation of ERK pathways plays an important role in the up-regulation of VEGF in conditions of serum starvation.10 p53, mutated in most colorectal cancers, was shown to be another regulator of VEGF expression. Cassano et al show that mutations of p53 induced VEGF expression in colorectal tumor samples, indicating that mutation of p53 may contribute to the angiogenic switch in colorectal cancer.11 The angiogenic switch of human colon cancer seems to occur simultaneously with the initiation of invasion. Takahashi et al reported a significant increase in vessel density and the expression of VEGF from Tis (tumor in situ) to T1 tumors, while adenomas showed the same VEGF values as Tis tumors.12 Other investigators reported that treatment with angiogenesis inhibitors, such as AMG-1470 or angiostatin, in the pre-malignant stage suppressed rapid tumor expansion and the progression to cancer.13 Aotake et al demonstrated the progression of angiogenesis as a step up in the carcinogenesis of human colorectal adenomas and carcinomas.14 In colorectal cancer COX-2 seems to play an important role in regulating tumor angiogenesis and tumor progression.15 Epidemiological studies have demonstrated a 40–50% decrease in the relative risk of colorectal cancer with the use of non-steroidal anti-inflammatory drugs (NSAIDs).16,17 COX-2 is regarded as an angiogenic factor in colorectal cancer. Modulation of VEGF by prostaglandins produced by COX-2 activity and/or induction of endothelial cell migration by COX-2 may contribute to tumor angiogenesis.18 Recently, Seno et al reported the importance of COX-2 and VEGF in the development of the large adenomas in adenomatous polyposis coli (APC)-mutated mice, and it was suggested that elevated prostaglandin E2, that stimulates its cell surface receptor subtype EP2, plays an important role in angiogenesis.19 JTE-522, a selective COX-2 inhibitor was shown to decrease the incidence of large adenomas, but not of small adenomas, through the inhibition of VEGF expression by interstitial cells.20 COX-2 in colorectal adenomas has been reported to be expressed more dominantly in interstitial cells than in epithelial cells.21 Sonoshita et al reported that the deficiency of either COX-2 or prostaglandin E2 receptor suppressed VEGF expression by interstitial cells in APC-mutated mice, and suggested that COX-2 played an important role in tumorigenesis and angiogenesis in adenomas of these mice.22 Hull et al also reported that COX-2 expression was localized to macrophages in the lamina propria and submucosa in the adenomas of APC-mutated mice.23 Seno et al observed that stromal expression of COX-2 in large adenomas was responsible for upregulation of VEGF that caused tumor angiogenesis.19 Sunayama et al showed that COX-2 participates strongly in polyp formation in APC-mutated mice. In this study, macrophages in the submucosal layer expressing COX-2 and VEGF were responsible for angiogenesis in large adenomas, and a selective COX-2 inhibitor reduced growth of adenoma mainly by its inhibitory effect on angiogenesis.20 Thus, the accumulation of macrophages expressing both VEGF and COX-2 in the submucosa of adenomas may be responsible for switching on angiogenesis and a prompt for unrestrained growth of adenomas.

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In colorectal cancer a wide range of molecules (e.g., HIF-1α, K-ras, p53, COX-2) regulate VEGF expression and thereby subsequent tumor angiogenesis and tumor progression. Presumably, targeting one of these individual pathways is not sufficient to control colorectal cancer. However further knowledge of the regulatory events involved will help to design “intelligent” and better therapies in future.

Prognostic Value of VEGF in Colorectal Cancer Patients The significance of angiogenesis as a prognostic marker has been investigated in many human tumors including colorectal cancer. Pro-angiogenic factors are already surrogate markers for the outcome of different anti-angiogenic treatment strategies. VEGF, as an important regulator of tumor angiogenesis, has been studied as an index of angiogenesis. In contrast to the number of studies reporting an association between high microvessel density and a greater incidence of metastases and decreased survival,3 few studies on the role of VEGF expression in predicting the prognosis of the patients with cancer, especially colorectal cancers, have been published. Amongst those studies some have indicated that VEGF expression is an independent factor in predicting patient prognosis,24-26 whilst others reported no such association.27-29 From those who did identify VEGF as a prognostic factor in colorectal cancer Akbulut et al could show in 52 consecutive colorectal patients with stage I to IV disease that high serumVEGF (S-VEGF) levels (> 5ng/ml per 1,000,000 platelets) correlated with poor patient survival (p=0.033).24 The prognostic significance in this study was further enhanced when unfavorable increased serum VEGF levels were combined with decreased nitric oxide to generate a new angiogenic index (p=0.002). In a larger study including 91 healthy volunteers and 614 patients scheduled to undergo resection for primary colorectal cancer, Werther et al showed a significant reduced overall survival in patients with S-VEGF levels over 465 pg/ml (95th percentile of S-VEGF of healthy individuals (p< 0.0001)) compared to patients with S-VEGF values below this value.26 Similarly, VEGF measurements from the tumor-draining mesenteric vein in 29 patients with or without metastatic disease revealed substantially higher VEGF levels in patients with distant metastases.30 However, in an immunohistochemistry based study in 145 colorectal carcinomas by Lee et al, VEGF expression within the tumor did not emerge as an independent risk factor in a multivariate analysis.28 Amongst 90 patients with non-metastatic colorectal cancer, VEGF expression was observed in 43 (48%) cases, whilst 29 of the 55 patients (53%) with metastases expressed the angiogenic factor. Khorana et al showed in a study with 131 consecutive Stage II and III colon cancer patients that VEGF expression in tumors was not significantly associated with survival.27 Surprisingly, a greater than twofold increase in median survival was associated with the presence of VEGF expressing tumor-associated macrophages in the stroma of tumors. Another study showed that metastatic colon cancers were associated with the simultaneous expression of the VEGF121, VEGF165, and VEGF189 splice variants in the primary tumors.31 In conclusion, VEGF serum levels are generally reported to correlate with the tumor burden and seem to correlate with the overall survival of patients with colorectal cancer. Studies using VEGF immunohistochemistry in tumor samples are less clear in terms of the prognostic value of VEGF. These confusing results may indicate that other pro-angiogenic factors have to be considered when establishing surrogate markers for metastatic disease. Posttranslational modifications of the different VEGF subtypes may also play a role in this context. With the increasing use of anti-angiogenic treatment options, the need for these kinds of markers will emerge but further research is needed to validate VEGF as a tumor (surrogate) marker in colorectal cancer.

The Role of VEGF in Metastasis of Colorectal Cancer Angiogenesis is required in two steps of the metastatic process: first, metastatic cells are only shed into the circulation if the tumor has been neovascularized. Second, the growth of macro-

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scopically detectable distant metastases at the target site again requires neovascularization.32 Micrometastases adopt a state of dormancy when lacking appropriate growth stimuli including pro-angiogenic signals. The importance of VEGF, amongst other pro-angiogenic molecules, in the metastatic process has been elucidated by many experimental and clinical studies. The development of liver metastases in colorectal cancer has been shown to be dependent on the expression of VEGF in tumor cells and the expression of the VEGFR-2 (KDR/Flk-1) and VEGFR-1(Flt1) receptors in tumor endothelium.33 In mice treated with monoclonal anti-VEGF antibody, a marked reduction in the number and size of liver metastases was observed as well as a reduction in the number of tumor blood vessels and a down-regulation of the KDR/flk-1 receptor.33 These data are supported by the results of Takahashi et al using microvessel quantification in metastatic and nonmetastatic tumor specimens. In this study, VEGF was significantly increased in metastatic colon cancer, correlating with an increase in neovascularization and KDR/flk-1 receptor expression. 3 More recently, transfection experiments using a human colon cancer cell line expressing high levels of VEGF mRNA and protein, demonstrated the involvement of VEGF in the metastatic process in vivo more directly, showing significantly enhanced development of liver metastases after intrasplenic injection in nude mice, compared to vector controls. In accordance with the concept of promotion of metastatic growth by induction of neovascularization, VEGF-transfected tumor cells did not show enhanced proliferation in vitro.34 As VEGF is a major contributor to metastatic neovascularization, different experimental strategies to antagonize VEGF-mediated effects have been developed. Similar to the use of neutralizing anti-VEGF monoclonal antibodies,33,35,36 experiments using a monoclonal anti-VEGFR-2 (KDR/Flk-1) antibody showed similar growth inhibition in terms of number and size of metastases and also resulted in tumor endothelial cell death in anti-VEGFR-2 treated mice, indicating a role for VEGF signalling as a survival factor for endothelial cells in colorectal liver metastases.37 In a different approach, the direct inhibition of the KDR/Flk-1 receptor by a specific tyrosine kinase inhibitor (SU5416) has been shown to inhibit metastasis formation and to lead to tumor endothelial cell apoptosis in an experimental colon cancer model, contributing to the concept of VEGF-induced endothelial cell survival.38 A colon cancer model interfering with VEGFR-1 and VEGFR-2 mRNA expression by ribozymes targeting the receptor mRNA yielded similar results.39 Recently, downstream inhibition of VEGF signalling using the mammalian target of rapamycin (mTOR) inhibitor, Rapamycin, prevented liver metastasis in a murine model of colon cancer.40 This approach might be promising, as it may not only interfere with VEGF signalling but could also inhibit the signalling pathways of other pro-angiogenic factors as well as altering cell cycle progression in tumor cells. COX-2 expression also seems to play a major role in colorectal cancer metastasis. Several studies showed a reduction of metastatic growth in human colorectal cancer xenografts in mice in response to COX-2 inhibition. While the selective COX-2 inhibitor, JTE-522, induced a marked decrease of liver metastases using a highly metastatic human xenograft, it did not inhibit subcutaneous tumor growth. In this study, VEGF expression was suppressed by JTE-522.15 Decreased VEGF expression in primary tumors accompanied by a reduction in the number of liver metastases was also shown when rofecoxib was used in a metastatic model using intrasplenic injection of tumor cells.41 In another study, however, JTE-522 did not affect VEGF production by tumor cells, yet an inhibition of liver metastases was shown.42 Apparently, the anti-metastatic effects of COX-2 inhibitors are not only mediated via VEGF, but other factors promoting invasion and proliferation such as platelet derived growth factor (PDGF) and matrix metalloproteinase (MMP-2). Further studies are needed to clarify the contribution of these factors to COX-2 induced tumor growth. Taken together, these experimental data indicate a vital role for VEGF in the development of colorectal liver metastases and may provide strategies to treat patients with metastatic disease.

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Table 1. Clinical colorectal cancer trials with drugs targeting the VEGF pathway Drug

Mechanism

Clinical Trails

Company

Bevacizumab (Avastin) ZD 6474 ZD 1839 (Iressa) ZK-222584 Angiozyme C 225 SU 5416 (Semaxanib) SU 6668

Human anti-VEGF Antibody VEGFR-2 tyrosine kinase inhibitor VEGF pathway inhibitor Targets VEGFR-1 Targets VEGFR-1 VEGF pathway inhibitor VEGFR-2 tyrosine kinase inhibitor Blocks VEGF, FGF and EGF receptor signalling mTOR inibition, Inhibits VEGF signalling Human anti-VEGFR-2 Antibody

Phase III Phase II Approved Phase II Phase II Phase II/III Phase III Phase II

Genentech AstraZeneca AstraZeneca Novartis Ribozyme Merck Pharmacia Sugen

Phase I

Wyeth

Phase I

ImClone

Rapamycin IMC-1C11

Recently, the VEGF family members VEGF-C and VEGF-D, ligands for the VEGF receptor-3 (VEGFR-3/Flt-4) have been associated with the lymphatic spread of malignant tumors. In two studies, VEGF-C mRNA levels correlated with lymphatic invasion and lymph node metastasis.43,44 Another study showed a correlation between VEGF-D expression and lymphatic metastasis and patient survival in Dukes stage A-C colorectal cancer. In this study, VEGF-D expression was found to be an independent prognostic factor for both disease free survival and overall survival.45 These intriguing findings might lead to a better understanding of the lymphatic spread of colorectal cancer and may be helpful in the design of drugs directed specifically against the lymphangiogenic VEGF subtypes and their receptors.

Anti-Angiogenic Therapy by Interference with the VEGF Pathway in Colorectal Cancer Angiogenesis has been shown to be critically involved in tumor development and progression in most solid tumors that have been studied. As an anti-cancer strategy, targeting tumor-associated vascular endothelial cells instead of the tumor itself has several theoretical advantages over common cytotoxic regimes (e.g., 5-fluorouracil (5-FU), oxaliplatin). The blockade of VEGF signalling may be a major part of future strategies for colorectal cancer. Among the approaches that have been proposed for blocking VEGF-induced endothelial cell proliferation and subsequent tumor angiogenesis, neutralizing anti-VEGF antibodies, blocking antibodies against the VEGFR-2 or selective inhibitors of the VEGFR-2 tyrosine kinase and VEGF pathway inhibitors, are currently in preclinical or clinical development. (see Table 1) We have also shown that other substances like the mTOR Inhibitor Rapamycin can inhibit downstream signalling of VEGF.40 Of all the anti-angiogenic drugs currently in clinical trials, Avastatin/Bevacizumab and Semaxanib/SU5416 are most developed and may be approved for use in colorectal cancer patients in the near future. Avastatin/Bevacizumab (Genentech) is a recombinant humanized monoclonal antibody to VEGF that is being incorporated into the front-line therapy for metastatic colorectal cancer. Preliminary results of a Phase II trial have been recently reported.46 Bevacizumab was combined with irinotecan (CPT-11), 5-FU and leucovorin in patients with previously untreated metastatic colorectal cancer. The main toxicity observed was an increased incidence

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of vascular events, both hemorrhagic and thrombotic. More recently encouraging results of a phase III trial of bevacizumab in combination with bolus irinotecan, 5-FU, leucovorin (IFL) as first-line therapy in subjects with metastatic colorectal cancer was presented at the ASCO meeting 2003. The addition of Bevacizumab to bolus-IFL chemotherapy resulted in a significantly increased survival (20.3 vs. 15.6 month), progression free survival (10.6 vs. 6.24 month), response rate (45% vs. 35%) and duration of response (10.4 vs. 7.1 month) as compared with bolus-IFL chemotherapy alone. The above mentioned side effects identified in the Phase II trial as possible safety issues were not observed in this recent trial. Semaxanib/SU5416 (Pharmacia) is a small-molecule receptor tyrosine kinase inhibitor that targets VEGFR-2. In a Phase I/II study of 27 evaluable patients treated with SU5416 in combination with 5-FU and leucovorin, 37 percent of patients had a complete or partial response to treatment as patients’ tumors were reduced by greater than 50 percent of their original size. Forty-four percent of patients had stable disease, meaning tumors were unchanged having neither grown nor reduced in size. Only seven percent of patients showed no response to the treatment. Among this group of patients with previously untreated advanced cancer, tumor growth and spread was delayed for a median of 9.0 months. Twenty-one of the 27 patients were still alive, so median survival data was unavailable. (May 2000 American Society of Clinical Oncology meeting)47,48 (Reviewed in ref. 49). Recent studies of the neutralizing antibody bevacizumab, and small molecule tyrosine kinase inhibitor SU5416, demonstrate that, while unlikely to be effective as monotherapy, incorporation of VEGF blockade into cytotoxic regimens (e.g., 5-FU) may increase overall response rates. However, incorporation may also produce new toxicities, including thromboembolic complications and bleeding. Newer oral agents, such as SU6668, ZD6474 and Rapamycin, are particularly attractive due to their potential for chronic therapy. Rapamycin (Wyeth) and its derivatives, CCI-779 (Wyeth) and RAD-001 (Novartis), bind to a highly conserved cellular protein FK-binding-protein (FKBP12) and the Rapamycin/ FKBP12 complex targets and inactivates mTOR, considered a master switch for cell cycle progression. In preclinical studies, mTOR inhibitors potently suppress growth and proliferation of numerous tumor cell lines in culture or when grown in mice as xenografts. CCI-779 and RAD-001 are being developed as anti-tumor drugs and are undergoing clinical trials. United States Federal Food and Drug Administration (FDA) has already approved Rapamycin for use in organ transplantation in conjunction with cyclosporine. Clinically, CCI-779 has shown evidence of anti-tumor activity but induced relatively mild side effects in patients. We have recently shown that Rapamycin may reduce the risk of cancer development while simultaneously providing effective immunosuppression. Experimentally, Rapamycin inhibited metastatic tumor growth and angiogenesis in in vivo mouse models of colon cancer. In addition, normal immunosuppressive doses of Rapamycin effectively controlled the growth of established tumors. From a mechanistic perspective, Rapamycin showed anti-angiogenic activity linked to decreased VEGF production and to a markedly inhibited response of vascular endothelial cells to stimulation by VEGF.40 Promising preclinical data illustrate the potential of these agents for tumor growth inhibition and even tumor regression, yet translation of novel therapeutics targeting the VEGF pathway into the clinic has proved a substantial challenge in itself. While showing clear evidence of anti-tumor activity over a broad spectrum of experimental colorectal cancer, the proper selection, dose, timing and sequence of anti-VEGF treatment in human cancer is not clear. Classic Phase I dose escalation trial design may need to be modified, as higher doses may not be optimal in all patients or for all tumors. In addition, alternate or secondary biological end points (e.g., non-progression) may be needed for early phase studies to document true activity, so as not to abandon effective agents. Future clinical trials are likely to build on past experience with stricter entry criteria, supportive care guidelines and the use of surrogate markers.

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References 1. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol 2001; 2:533-543. 2. Steinbach G, Lynch PM, Phillips RK et al. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med 2000; 342:1946-1952. 3. Takahashi Y, Kitadai Y, Bucana CD et al. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 1995; 55:3964-3968. 4. Ono T, Miki C. Factors influencing tissue concentration of vascular endothelial growth factor in colorectal carcinoma. Am J Gastroenterol 2000; 95:1062-1067. 5. Wong MP, Cheung N, Yuen ST et al. Vascular endothelial growth factor is up-regulated in the early pre-malignant stage of colorectal tumor progression. Int J Cancer 1999; 81:845-850. 6. Forsythe JA, Jiang BH, Iyer NV et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996; 16:4604-4613. 7. Rak J, Mitsuhashi Y, Bayko L et al. Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. Cancer Res 1995; 55:4575-4580. 8. Arbiser JL, Moses MA, Fernandez CA et al. Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc Natl Acad Sci U S A 1997; 94:861-866. 9. Milanini J, Vinals F, Pouyssegur J et al. p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts. J Biol Chem 1998; 273:18165-18172. 10. Jung YD, Nakano K, Liu W et al. Extracellular signal-regulated kinase activation is required for up-regulation of vascular endothelial growth factor by serum starvation in human colon carcinoma cells. Cancer Res 1999; 59:4804-4807. 11. Cassano A, Bagala C, Battelli C et al. Expression of vascular endothelial growth factor, mitogen-activated protein kinase and p53 in human colorectal cancer. Anticancer Res 2002; 22:2179-2184. 12. Takahashi Y, Ellis LM, Mai M. The angiogenic switch of human colon cancer occurs simultaneous to initiation of invasion. Oncol Rep 2003; 10:9-13. 13. Bergers G, Javaherian K, Lo KM et al. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science 1999; 284:808-812. 14. Aotake T, Lu CD, Chiba Y et al. Changes of angiogenesis and tumor cell apoptosis during colorectal carcinogenesis. Clin Cancer Res 1999; 5:135-142. 15. Yamauchi T, Watanabe M, Hasegawa H et al. The potential for a selective cyclooxygenase-2 inhibitor in the prevention of liver metastasis in human colorectal cancer. Anticancer Res 2003; 23:245-249. 16. DuBois RN, Smalley WE. Cyclooxygenase, NSAIDs, and colorectal cancer. J Gastroenterol 1996; 31:898-906. 17. Thun MJ, Namboodiri MM, Heath CW, Jr. Aspirin use and reduced risk of fatal colon cancer. N Engl J Med 1991; 325:1593-1596. 18. Tsujii M, Kawano S, Tsuji S et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998; 93:705-716. 19. Seno H, Oshima M, Ishikawa TO et al. Cyclooxygenase-2 and prostaglandin E(2) receptor EP(2)-dependent angiogenesis in Apc(Delta716) mouse intestinal polyps. Cancer Res 2002; 62:506-511. 20. Sunayama K, Konno H, Nakamura T et al. The role of cyclooxygenase-2 (COX-2) in two different morphological stages of intestinal polyps in APC(Delta474) knockout mice. Carcinogenesis 2002; 23:1351-1359. 21. Bamba H, Ota S, Kato A et al. High expression of cyclooxygenase-2 in macrophages of human colonic adenoma. Int J Cancer 1999; 83:470-475. 22. Sonoshita M, Takaku K, Sasaki N et al. Acceleration of intestinal polyposis through prostaglandin receptor EP2 in APC(Delta 716) knockout mice. Nat Med 2001; 7:1048-1051. 23. Hull MA, Booth JK, Tisbury A et al. Cyclooxygenase 2 is up-regulated and localized to macrophages in the intestine of Min mice. Br J Cancer 1999; 79:1399-1405. 24. Akbulut H, Altuntas F, Akbulut KG et al. Prognostic role of serum vascular endothelial growth factor, basic fibroblast growth factor and nitric oxide in patients with colorectal carcinoma. Cytokine 2002; 20:184-190. 25. Kang SM, Maeda K, Onoda N et al. Combined analysis of p53 and vascular endothelial growth factor expression in colorectal carcinoma for determination of tumor vascularity and liver metastasis. Int J Cancer 1997; 74:502-507.

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26. Werther K, Christensen IJ, Brunner N et al. Soluble vascular endothelial growth factor levels in patients with primary colorectal carcinoma. The Danish RANX05 Colorectal Cancer Study Group. Eur J Surg Oncol 2000; 26:657-662. 27. Khorana AA, Ryan CK, Cox C et al. Vascular endothelial growth factor, CD68, and epidermal growth factor receptor expression and survival in patients with Stage II and Stage III colon carcinoma: a role for the host response in prognosis. Cancer 2003; 97:960-968. 28. Lee JC, Chow NH, Wang ST et al. Prognostic value of vascular endothelial growth factor expression in colorectal cancer patients. Eur J Cancer 2000; 36:748-753. 29. Takahashi Y, Tucker SL, Kitadai Y et al. Vessel counts and expression of vascular endothelial growth factor as prognostic factors in node-negative colon cancer. Arch Surg 1997; 132:541-546. 30. Gunsilius E, Tschmelitsch J, Eberwein M et al. In vivo release of vascular endothelial growth factor from colorectal carcinomas. Oncology 2002; 62:313-317. 31. Tokunaga T, Oshika Y, Abe Y et al. Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer. Br J Cancer 1998; 77:998-1002. 32. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995; 1:27-31. 33. Warren RS, Yuan H, Matli MR et al. Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis. J Clin Invest 1995; 95:1789-1797. 34. Kondo Y, Arii S, Mori A et al. Enhancement of angiogenesis, tumor growth, and metastasis by transfection of vascular endothelial growth factor into LoVo human colon cancer cell line. Clin Cancer Res 2000; 6:622-630. 35. Okamoto K, Oshika Y, Fukushima Y et al. Inhibition of liver metastasis of colon cancer by in vivo administration of anti-vascular endothelial growth factor antibody. Oncol Rep 1999; 6:553-556. 36. Kanai T, Konno H, Tanaka T et al. Anti-tumor and anti-metastatic effects of humanvascular-endothelial-growth-factor-neutralizing antibody on human colon and gastric carcinoma xenotransplanted orthotopically into nude mice. Int J Cancer 1998; 77:933-936. 37. Bruns CJ, Liu W, Davis DW et al. Vascular endothelial growth factor is an in vivo survival factor for tumor endothelium in a murine model of colorectal carcinoma liver metastases. Cancer 2000; 89:488-99. 38. Shaheen RM, Davis DW, Liu W et al. Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis. Cancer Res 1999; 59:5412-5416. 39. Pavco PA, Bouhana KS, Gallegos AM et al. Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors. Clin Cancer Res 2000; 6:2094-2103. 40. Guba M, von Breitenbuch P, Steinbauer M et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002; 8:128-35. 41. Yao M, Kargman S, Lam EC et al. Inhibition of cyclooxygenase-2 by rofecoxib attenuates the growth and metastatic potential of colorectal carcinoma in mice. Cancer Res 2003; 63:586-592. 42. Nagatsuka I, Yamada N, Shimizu S et al. Inhibitory effect of a selective cyclooxygenase-2 inhibitor on liver metastasis of colon cancer. Int J Cancer 2002; 100:515-519. 43. Kawakami M, Furuhata T, Kimura Y et al. Quantification of vascular endothelial growth factor-C and its receptor-3 messenger RNA with real-time quantitative polymerase chain reaction as a predictor of lymph node metastasis in human colorectal cancer. Surgery 2003; 133:300-308. 44. Akagi K, Ikeda Y, Miyazaki M et al. Vascular endothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues. Br J Cancer 2000; 83:887-891. 45. White JD, Hewett PW, Kosuge D et al. Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma. Cancer Res 2002; 62:1669-1675. 46. Kabbinavar F, Hurwitz HI, Fehrenbacher L et al. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003; 21:60-65. 47. Kuenen BC, Rosen L, Smit EF et al. Dose-finding and pharmacokinetic study of cisplatin, gemcitabine, and SU5416 in patients with solid tumors. J Clin Oncol 2002; 20(6):1657-1667. 48. Stopeck A, Sheldon M, Vahedian M et al. Results of a Phase I dose-escalating study of the antiangiogenic agent, SU5416, in patients with advanced malignancies. Clin Cancer Res 2002; 8(9):2798-2805. 49. Berlin JD. Targeting vascular endothelial growth factor in colorectal cancer. Oncology (Huntingt) 2002; 16(8 Suppl 7):13-15.

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CHAPTER 9

Vascular Endothelial Growth Factor in Malignant Disease of the Central Nervous System David Stefanik

Abstract

V

ascular Endothelial Growth Factor (VEGF) is a major contributor to the growth of malignant tumors of the central nervous system. It stimulates tumor angiogenesis and vascular proliferation characteristic of high grade gliomas. Elevated expression of VEGF is one the factors responsible for the virulent nature of these tumors. The production of VEGF by malignant glial cells in response to ionizing radiation contributes to treatment failure. The rat C6 glioma is similar to human gliomas with respect to VEGF pathophysiology. Interruption of VEGF-Receptor signaling in preclinical models effectively suppresses tumor growth and demonstrates the potential for anti-angiogenic therapy.

VEGF in the Normal Brain Intact VEGF-Receptor signaling is required for maturation of the central nervous system (CNS). Mutant mice heterozygous for VEGF die in utero and develop multiple anomalies including failure of vascularization of the neuroepithelium, disorganization of neuroepithelial cells, and underdevelopment of the forebrain.1 A single mutant allele can bring this about. In mature brain tissue VEGF is distributed in areas surrounding the microvasculature where it may assist in maintaining the differentiated state.2 VEGF is also produced in response to CNS trauma. In response to cold thermal injury, VEGF isoform A is upregulated in astrocytes, inflammatory cells, and neovascular endothelium in the rat brain. Increased production of VEGF mRNA was demonstrated as early as six hours after injury by in situ hybridization.3

VEGF Is Upregulated in Malignant Disease of the CNS Human brain tumors comprise a group of diseases which vary in their natural history, histopathology, and response to treatment. Heterogeneity also exists within each histopathologic category of tumor. In adults, the most common tumors arise from cells which comprise the supportive stroma of the CNS.4 Collectively, they are called gliomas. These include astrocytomas, oligodendrogliomas, and ependymomas. Up-regulation of VEGF and its receptors has been demonstrated in each.5-7 Astrocytomas are the most common glial tumors. They are subdivided on the basis of how undifferentiated the tissue appears microscopically. Grade I tumors bear the closest resemblance to normal brain. Grade IV are the most undifferentiated. Low grade astrocytomas progress more slowly than their high grade counterparts whose growth can be explosive. Grade IV astrocytoma is also known as glioblastoma multiforme, one of the deadliest of all malignant diseases. Figure 1 is a CT (computerized tomography) scan of a VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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Figure 1. Contrast enhancement: the signature of VEGF. Iodinated molecules of high molecular weight pool in the perivascular space due to capillary leakage. A typical CT scan for high grade glioma at the time of presentation or upon disease recurrence.

patient with glioblastoma multiforme. It demonstrates a large parietal lobe mass which “enhances” after the intravenous administration of iodinated molecules of high molecular weight. The iodinated reagent accumulates in the perivascular space around leaky capillaries allowing the vascular rim of the tumor to stand out in contrast to other tissue. A region of hypodensity indicative of edema surrounds the area of enhancement. While multiple growth factors are potentially operative in glioma angiogenesis, only VEGF is known to induce vascular permeability. Angiogenesis increases markedly from the low grade tumors (grades I and II) to the high grade lesions (grades III and IV). VEGF and its receptors are upregulated in most, but not all, astrocytomas.8 Production of VEGF parallels the angiogenic phenotype, with high grade tumors being more likely to produce VEGF. Furthermore, low grade astrocytomas which produce VEGF have the same dismal prognosis as high grade lesions.9 Elevated VEGF production has also been demonstrated in CNS tumors of non glial origin. These include craniopharyngiomas, meningiomas, pituitary adenomas, and hemangioblastomas.10-13 In these nonglial tumors, VEGF production is variable. VEGF is elevated in virtually all hemangioblastomas. In meningioma VEGF content correlates with the degree of differentiation. The most undifferentiated lesions produce the greatest amount of VEGF. However, Lamszus et al found no correlation between VEGF content and vascularity or invasiveness.11 Taken together these findings suggest that VEGF is a factor in the evolution of nonglial tu-

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mors, but not the only one. Of historical note, an early isolate of VEGF came from cultured pituitary folliculostellate cells.14 Tumors which metastasize to the brain also produce VEGF.15 Fortunately, brain tumors are rare in children. The majority of CNS tumors in children arise from astrocytes ( astrocytomas) or from the granular layer of the cerebellum (medulloblastomas). Both are associated with enhanced VEGF production.16,17 Gliomas are the most common malignant tumors of the central nervous system. They have been studied extensively with regard to VEGF. The remaining discussion will focus on them.

Angiogenesis in Glioma One of the major differentiating features between low grade and high grade gliomas is the degree of vascularity. The progression to high grade is characterized by increased vascularity and the presence of endothelial cell proliferation. Swollen endothelial cells become prominent, and abnormal vascular channels resembling glomerular structures develop in the most undifferentiated lesions. Neoangiogenesis in human glioma is driven by multiple molecules including VEGF, angiopoietins, fibroblast growth factors, platelet derived growth factor, epidermal growth factor, and transforming growth factors.18 Macrophages, themselves capable of releasing multiple modulators of vascular remodeling including VEGF, play a role.19 Down-regulation of negative modulators of angiogenesis is also seen in the progression of glioma. The anti-angiogenic molecule, thrombospondin, is shut off in high grade lesions.20 Folkman has advanced the concept of the “angiogenic switch” in tumor progression. A major tenet is that neoangiogenesis is dependent upon the local balance between pro and anti-angiogenic forces.21 Gliomas, with multi-factorial pro and anti-angiogenic molecules, are a classical illustration of this concept. Aberrant VEGF production plays a significant role in the pathophysiology of glioma progression. VEGF is produced in at least four isoforms. Three isoforms, VEGF,121 VEGF165 and VEGF,189 have been demonstrated in glioblastoma multiforme.22 In situ hybridiziation demonstrates a progressive increase in VEGF mRNA from very low levels in normal brain to a fifty fold elevation in glioblastoma where expression is detected in palisading cells around areas of necrosis.23 Likewise, VEGF expression correlates strongly with neovascularization and tumor progression in oligodendroglioma.24 VEGF is produced by malignant glial cells which are distributed both centrally and peripherally within tumors.25,26 In situ hybridization experiments demonstrate mRNA for VEGF in malignant glial cells often juxtaposed to areas of necrosis. Cells producing VEGF also infiltrate the normal brain adjacent to tumor. The receptors for VEGF are up-regulated in the developing microvasculature of gliomas.27-29 Increased expression of mRNA for VEGFR-1 (Flt-1, fms-like tyrosine kinase) and VEGFR-2 (Flk-1/KDR, Fetal liver kinase 1, murine homologue of human Kinase insert Domain-containing Receptor) is found in endothelial cells of the tumor neovasculature and in normal brain adjacent to tumors but not in the established vasculature of the normal brain. VEGF can be demonstrated on or within the newly developing blood vessels. Taken together, these findings support the hypothesis that VEGF is produced and secreted by malignant glial cells and is then sequestered by receptors on activated endothelium. Figure 2 is an immunohistochemical evaluation of human glioblastoma which demonstrates VEGF in the glial cells surrounding capillaries and in the capillary walls themselves. VEGFR-1 (Flt-1) is also present on macrophages. Recruitment of macrophages is mediated in part by VEGF-Flt-1 receptor signaling.30 The presence of macrophages further contributes to neoangiogenesis and correlates with poor prognosis in malignant tumors of the central nervous system.31 VEGF is one of a number of growth factors capable of stimulating endothelial cell proliferation in brain tumors.18 Angiopoietin-Tie2/Tek receptor signaling is an important early event along the angiogenic pathway.32 Vascular remodeling in glioma is dependent upon signals generated by both VEGF and the angiopoietins. When glioma cells are implanted into the brains

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Figure 2. Immunohistochemistry of glioblastoma multiforme: the brown stain indicates the presence of VEGF in malignant glial cells surrounding “glomeruloid” vessels. VEGF is also present in the endothelial cells themselves.

of rats or mice, tumor “take” is facilitated by co-option of existing vessels.33 The co-opted vasculature initially regresses under the influence of angiopoietin-2. Tumor cell death occurs in areas of central necrosis. VEGF becomes upregulated in peripheral regions in response to hypoxia. Consequently, a robust angiogenic response occurs in the peripheral rim. In their elegant work, Holash et al studied the co-ordinated expression of Angiopoietin-2 and VEGF in specimens of C6 gliomas as well as human glioblastoma.33 They identified Angiopoietin-2 as an early signal which destabilizes vascular endothelium. The coincident expression of Angiopoietin-2 and VEGF produces neoangiogenesis. Angiopoietin-2 expression in the absence of VEGF leads to endothelial cell death and vascular regression. Other studies have documented the involvement of PDGF (platelet derived growth factor), the FGF’s (fibroblast growth factors), and EGF (epidermal growth factor) in glioma angiogenesis.34-36 The picture which emerges has VEGF as a major contributor to angiogenesis in malignant disease of the CNS albeit with co-ordinated input from a number of other growth factors. Integrins present on the surface of endothelial cells are important for cell to cell signaling and attachment to the extracellular matrix. Friedlander et al have identified two separate angiogenic pathways.37 VEGF drives a pathway in which alphavbeta5 integrin is up-regulated whereas bFGF (basic fibroblast growth factor) drives a pathway mediated by alphavbeta3 integrin. In situ hybridization of glioblastoma demonstrates mRNA for both beta3 and beta5 subunits in contrast to normal brain microvasculature where neither was present.38 These findings support the concept that VEGF and bFGF both contribute to angiogenesis in high grade glioma.

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Factors Influencing VEGF Production VEGF exists in four major isoforms of 121, 165, 189, and 206 amino acids. The isoforms are created by alternative splicing of a single gene found on chromosome six in humans.39 VEGF165, the predominant species, is a weakly acidic heparin binding protein. It is present on cell surfaces and secreted. VEGF121 is the most soluble. VEGF189 and VEGF206 bind more avidly to heparin and are sequestered in the extracellular matrix. Proteolytic cleavage of the longer isoforms by plasmin produces biologically active fragments which contribute to angiogenesis. The gene for VEGF is regulated by hypoxia. The promoter region contains a 28 base pair sequence under control of the transcriptional activator, Hypoxia Inducible Factor I (HIF-1).40 VEGF expression is increased in response to hypoxia in cultured cells of human gliomas.23 Post transcriptional stabilization of mRNA coding for VEGF also contributes to elevated VEGF concentrations in response to hypoxia.41 In situ hybridization of glioblastoma multiforme demonstrates the presence of VEGF producing cells in close proximity to necrotic (hypoxic) regions.23 Evidence from multiple sources points to the importance of hypoxic up-regulation of VEGF expression in malignant disease of the CNS. Acidosis also contributes to VEGF production independently of hypoxia.42 Fukumura et al used fluorescence ratio imaging microscopy to evaluate VEGF production in human gliomas growing in a mouse cranial window. They mapped out regions of VEGF production in relation to the pO2 and pH of the tissue. The highest levels of VEGF were found in acidotic regions independent of the degree of local oxygenation. They concluded that VEGF is up-regulated by hypoxia and acidosis via different mechanisms.42 Growth factors including bFGF, PDGF-BB, and EGF stimulate VEGF production by cultured glioma cell lines.43 Stimulation of the VEGF promotor by activation of the EGF Receptor occurs under hypoxic and normoxic conditions.44 Activation of oncogene pathways can stimulate VEGF production. Src stimulates VEGF production.45 Ras up-regulates VEGF under both normoxic and hypoxic conditions.46 Inhibition of the ras pathway by genetic or pharmacologic manipulation results in decreased VEGF expression.47 Wild type p53 suppresses VEGF expression.45 Malignant progression in glioma is associated with loss of function of the tumor suppressor gene p16. Restoration of p16 activity inhibits VEGF expression and angiogenesis.48 In hemangioblastomas the von Hippel-Lindau (VHL) tumor suppressor gene is mutated.49 The function of wild type (normal) VHL is to dampen expression of hypoxia inducible genes including HIF-1. Hemangioblastomas, as their name suggests, are highly vascular tumors which express high levels of VEGF. Radiation stimulates VEGF expression in cultured glioma cells. VEGF was elevated in the conditioned media of human glioblastoma T98 and U87 cells following radiation exposure.50 The underlying mechanism involves activation of the Mitogen Activated Protein Kinase (MAPK) pathway. Ionizing radiation stimulated the MAPK pathway in both primary and malignant astrocytes of rat origin.51 MAPK activation was most pronounced in malignant cells, and multiple doses of radiation had a greater effect than single doses.

C6 Glioma Is an Excellent Model for the Study of High Grade Human Glioma with Regard to VEGF C6 glioma is a highly vascular tumor of rat origin similar in histologic appearance to human high grade glioma.52 “Palisading cells” producing VEGF surround areas of necrosis in both. As in human tumors, VEGF receptors are up-regulated on endothelial cells within tumor and at the interface between tumor and normal brain but not in the normal brain. Hypoxia stimulates VEGF production by C6 cells, and mRNA encoding VEGF is stabilized post transcriptionally.53 Similar to human gliomas, angiogenesis in C6 tumors depends upon the interaction between VEGF and angiopoietin-2.33,54 Angiogenesis in C6 tumors is dependent on VEGF production. VEGF withdrawal leads to endothelial cell apoptosis and secondary tumor necrosis.55

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VEGF Is Responsible for the Virulent Nature of High Grade Gliomas While multiple growth factors influence angiogenesis in glioma, none is more crucial to the process than VEGF. Glial cell lines vary in their production of VEGF, and tumorigenicity correlates with VEGF production.56 Furthermore, cell lines of low virulence can be converted to high virulence by engineering them to express more VEGF.57 Studies where cells are “re-engineered” with respect to VEGF-receptor signaling clearly demonstrate that glioma growth and VEGF signaling go hand in hand. Benjamin and Keshet transfected C6 cells with a plasmid expressing VEGF under control of a tetracycline sensitive promotor.55 Tetracycline inhibited the promotor, and production of VEGF was repressed by the addition of tetracycline to the animals’ drinking water. C6 tumors were allowed to grow to as large as one centimeter in diameter when VEGF was shut off. Microscopic evaluation of the VEGF deprived tumors demonstrated endothelial cell detachment and regression of both newly formed and mature vessels indicating that VEGF serves as a survival factor for established vasculature in this tumor model. Marked tumor necrosis was seen around areas of vascular involution. Cheng et al transfected the human glioblastoma line U87MG with an antisense construct of the gene expressing VEGF.58 The cells produced less VEGF but retained their usual growth characteristics in vitro. However, they exhibited diminished ability to stimulate the migration of endothelial cells. Untransfected wild type cells readily formed large tumors when inoculated in the flanks of immunocompromised mice. They also grew intracranially. The antisense transfected cells showed marked impairment of growth when implanted subcutaneously or intracranially. Microvascular density was diminished in the small tumors which developed from the antisense-transfected cell line. Transfected cells which spontaneously lost the antisense expression reverted to the highly malignant and vasculogenic phenotype. Similar results were obtained using antisense VEGF transfected C6 cells and by directly injecting established tumors with a recombinant adenovirus expressing antisense VEGF.59,60 Millauer et al created a retrovirus expressing a mutated form of VEGFR-2 (Flk-1).61 Cells infected by the virus produced receptors for VEGF which were defective in VEGF signal transduction but which retained normal signal transduction pathways for other growth factors such as PDGF. Such cells were under the influence of a “dominant negative Flk-1 mutant.” The growth of C6 cells was unaltered in vitro by exposure to conditioned media from cell lines producing the “dominant negative” retrovirus. However, C6 tumor growth was inhibited in vivo in nude mice when the tumors were exposed to the retrovirus expressing truncated Flk-1 either at the time of tumor inoculation or five days afterwards. Microscopic examination of the treated tumors revealed a central necrotic core surrounded by a rim of viable cells with fewer capillaries. The studies by Benjamin, Cheng, and Millauer clearly demonstrate that intact VEGF-receptor signaling is required for tumorgenicity in glioma.55,58,61 When otherwise virulent U87MG or C6 cells are re-engineered to produce less VEGF, they are no longer killers. Likewise, when the VEGFR-2 (Flk-1/KDR) receptor can be silenced by creating one that doesn’t function, angiogenesis and tumor growth is profoundly inhibited. All tumors contain multiple molecular derangements; none is more crucial to the growth of high grade glioma than overexpression of VEGF.

Interrupting VEGF-Receptor Signaling Inhibits Glioma Growth in Preclinical Models Glioma growth has been inhibited in preclinical models by strategies which target VEGF-receptor signaling in tumors which have not been re-engineered or epi-genetically modified. These include treatments with monoclonal antibodies directed against VEGF, monoclonal antibodies to VEGFR-1 and VEGFR-2, VEGF conjugated with diptheria toxin, soluble decoy receptors for VEGF (VEGF-Trap), and low molecular weight tyrosine kinase inhibitors (SU6668, SU5416).62-69

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Intra-peritoneal administration of the VEGF neutralizing antibody, 4.6.1, caused growth inhibition in a variety of human tumor xenografts including the G55 glioblastoma cell line.62 The antibody had no effect on cell proliferation in vitro. However, established G55 tumors treated in vivo with anti-VEGF had 20% of the weight of tumors treated with a control antibody. Microscopic evaluation showed a decrease in the vasculature of those tumors treated with 4.6.1. The antibody inhibited but did not eradicate the growth of G55 tumors. The authors speculated that additional angiogenic factors such as the Fibroblast Growth Factors might be responsible for growth. Yuan et al observed the effects of 4.6.1 on U87 growth in dorsal skinfold and cranial window chambers of mice.70 The antibody caused a reduction in vascular permeability within six hours of intravenous administration. The same effect was observed four days after intra-peritoneal administration of antibody. Other anti-VEGF antibodies besides 4.6.1 have slowed the growth of xenografted gliomas.63,65 Antibodies directed against both VEGFR-1 and VEGFR-2 have likewise demonstrated a capacity to impede the growth of gliomas. DC101 is a monoclonal antibody against VEGFR-2 which inhibited the growth of human GBM-18 tumors in a mouse xenograft.64 Regression of established glioblastomas was observed with DC101. The antibody inhibited tumor growth throughout the duration of treatment . However, progressive tumor growth took place upon cessation of treatment. Tumors treated with DC101 became pale and avascular.64 A monoclonal antibody to VEGFR-1 inhibited the growth of C6 tumors in mice.65 Tumors treated with the antibody to VEGFR-1 accumulated fewer macrophages in comparison to tumors treated with a control antibody. They also showed impaired vascular maturation. VEGF-Trap is a molecule designed as a “decoy receptor” for VEGF. It consists of portions of the receptors for VEGF fused to the constant (Fc) region of IgG1. As implied by its name, it traps VEGF before binding to true receptors can occur. VEGF-Trap effectively inhibited the growth of C6 tumors in mice.67 Neoangiogenesis was markedly inhibited, but some viable tumor cells survived by “co-opting” the underlying host microvasculature. SU6668 is a low molecular weight tyrosine kinase inhibitor capable of blocking receptors for VEGF, FGF, and PDGF.68 It can be administered either intra-peritoneally or orally. It demonstrated growth inhibition against a wide variety of tumors tested in a mouse xenograft. The A431 human epidermoid tumor was particularly susceptible with 97% growth inhibition. Half of all A431 tumors were permanently eradicated. SU6668 did well, albeit less spectacularly, against C6 tumors which showed 81% growth inhibition and the human glioma, SF763T, whose growth was inhibited by 79%. Intravital videomicroscopy revealed diminished tumor vessel density in C6 tumors treated with SU6668. However, even treated tumors showed an increase in vessel density from day 10 to day 22. Collectively, the preclinical studies targeting VEGF signaling share the following common denominators: (1) tumor growth is impeded, and the magnitude of the effect is impressive. However, gliomas are not completely eradicated in the process. (2) microvascular density is decreased; it is not eliminated. Following cessation of treatment, tumor growth resumes.

Current Therapy of Glioma: VEGF Contributes to Treatment Failure Treatment of glioma generally consists of subtotal resection followed by radiation therapy. Tumors infiltrate normal brain parenchyma, and total removal is rarely achievable. Fractionated radiation is delivered postoperatively to doses in the order of 6000 to 6500 cGy in daily fractions of 180-200 cGy. Chemotherapy is often given concurrent with and following radiation in high grade lesions. The prognosis depends largely upon the grade of tumor and the extent of resection.71,72 Grade I and II tumors have few mitotic figures, little cellular pleomorphism, and sparse normal appearing neovasculature. Median survival with grade I/II tumors is measured in years. Grade III/IV lesions are a different matter. These tumors have a high mitotic index, much cellular pleomorphism, and abundant abnormal appearing neovasculature. Despite the aggressive treatment outlined above, median survival is measured in months for high

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grade gliomas. The vast majority of recurrences happen “locally,” that is, within the original tumor volume. Autopsy specimens demonstrate VEGF expression in cells spread throughout the recurrent tumor and in normal-appearing astrocytes in the adjacent normal brain.73 Why does aggressive treatment fail to control high grade glioma? Cell lines derived from gliomas are not intrinsically “radioresistant.”74 They exhibit the same sensitivity to radiation as “radiocurable” cell lines in clonogenic assays. The answer may be that gliomas produce VEGF in response to ionizing radiation.50,51 Gorski et al demonstrated that VEGF is a survival factor which protects endothelial cells from the lethal effects of radiation in vitro.50 Conversely, blocking VEGF enhances the ability of radiation to kill endothelial cells. The authors put forth the hypothesis that “radiation resistance” is mediated by the protective effect of VEGF on the microvasculature. Geng et al validated this hypothesis by observing the growth of tumor vasculature in a vascular window model.75 They grew the murine glioma cell line, GL261, in the hindlimbs of mice and measured vascular growth in response to radiation alone or in combination with VEGF receptor blockade. They found that VEGF receptor blockade sensitized the endothelium to ionizing radiation and “eliminated the resistance phenotype.” Radiation has been combined with anti-VEGF approaches in the treatment of established glioma xenografts. Monoclonal antibodies to VEGF and VEGFR-2 have been employed in combination with radiation.49,76,77 The low molecular weight tyrosine kinase inhibitor, SU5416, has also been used with radiation.75 Combined therapy demonstrates improvement over radiation alone. The growth of tumors is delayed by combined therapy, however, eradication of tumors has not been reported.

Summary and Future Directions High grade gliomas are incurable by current methods of treatment. They possess the ability to regenerate by mounting a vigorous angiogenic response. VEGF is central to the process. It is the main “accelerant” which fuels tumor growth before and after conventional treatment. In preclinical models, blockade of VEGF-receptor signaling disrupts angiogenesis which causes tumor shrinkage and growth delay. The magnitude of the effect is impressive. However, secondary growth factors (FGF’s, PDGF, TGF etc) capable of stimulating angiogenesis are operative in high grade glioma. They can drive the angiogenic engine in the face of VEGF blockade. True believers (the author is one) in the concept of cancer control by blockade of tumor angiogenisis must understand that the process is multi-factorial. Strategies which complement VEGF blockade must be identified. Potential avenues of approach include: (1) Disrupting the process of “vascular co-option” whereby tumor cells engage the existing host vasculature. This phenomenon seems to be a common feature of early disease development as well as tumor regrowth following treatment. (2) Targeted therapy to disrupt signaling of the secondary growth factors. (3) Disruption of integrin signaling. It has been only two decades since the discovery that “tumor cells secrete a vascular permeability factor.”78 We know it now as Vascular Endothelial Growth Factor. Since then the central role of VEGF in the pathophysiology of malignant disease has been elucidated. Molecules capable of blocking its effects have gone from the preclinical to the clinical stage of investigation. Unfortunately, angiogenesis inhibitors have had a disappointing track record in early clinical trials. Large potentially unstable molecules have been used as single agents. Often, doses employed in human trials have been significantly smaller on a milligram for kilogram basis than those employed in mice. The “true believer” must not be deterred. We are looking for the right combination of anti-angiogenic molecules. Single agents won’t do the trick against a multi-factorial process. In the future, heretofore uncontrollable tumors such as glioblastoma will be cured by the delivery of a “cocktail” of angiogenesis inhibitors. The cocktail will include at least one potent blocker of VEGF-receptor signaling. Take it to the bank. Keep looking for the ingredients.

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References 1. Ferrara N, Carver-Moore K, Chen H et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996; 380:439-442. 2. Monacci W, Merrill M, Oldfield E. Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues. Am J Physiol 1993; 264:995-1002. 3. Nag S, Eskandarian MR, Davis J et al. Differential expression of vascular endothelial growth factor A (VEGF-A) and VEGF-B after brain injury. J Neuropath Exp Neurol 2002; 61:778-788. 4. De Girolami U, Frosch MP, Anthony DC. The Central Nervous System. In: Cotran RS, Kumar V, Robbins SL, eds. Pathologic Basis of Disease. 5th ed. Philadelphia: WB Saunders Co., 1994:1295-1356. 5. Plate KH, Breir G, Weich HA et al. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo. Nature 1992; 359:845-847. 6. Christov C, Adle-Biassette H, Guerinel C et al. Immunohistochemical detection of vascular endothelial growth factor (VEGF) in the vasculature of oligodendrogliomas. Neuropathol Appl Neurobiol 1998; 24:29-35. 7. Pietsch T, Valter MM, Wolf HK et al. Expression and distribution of vascular endothelial growth factor protein in human brain tumors. Acta Neuropathol 1997; 93:109-117. 8. Berkman RA, Merrill MJ, Reinhold WC et al. Expression of the vascular permeability factor/vascular endothelial growth factor gene in central nervous system neoplasms. J Clin Invest 1993; 91:153-159. 9. Yao Y, Kubota T, Sato K et al. Prognostic value of vascular endothelial growth factor and its receptors flt-1 and flk-1 in astrocytic tumors. Acta Neurochir (Wien) 2001; 143:159-166. 10. Vaquero J, Zurita M, Ke Oya S et al. Expression of vascular permeability factor in craniopharyngioma. J Neurosurg 1999; 91:831-834. 11. Lamaszus K, Lengler U, Schmidt NO et al. Vascular endothelial growth factor, hepatocyte growth factor/scatter factor, basic fibroblast growth factor, and placenta growth factor in human meningiomas and their relation to angiogenesis and malignancy. Neurosurgery 2000; 46:938-947. 12. Lloyd RV, Scheithauer BW, Kuroki T et al. Vascular endothelial growth factor (VEGF) expression in human pituitary adenomas and carcinomas. Endocr Pathol 1999; 10:229-235. 13. Wizigmann-Voss S, Breir G, Risau W et al. Up-regulation of vascular endothelial growth factor and its receptors in von Hippel-Lindau disease-associated and sporadic hemangioblastomas. Cancer Res 1995; 55:1358-1364. 14. Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculostellate cells. Proc Natl Acad Sci USA 1989; 86:7311-7315. 15. Stockhammer G, Obwegeser A, Kostron H et al. Vascular endothelial growth factor (VEGF) is elevated in brain tumor cysts and correlates with tumor progression. Acta Neuropathol (Berl) 2000; 100:101-105. 16. Huber H, Eggert A, Janss R et al. Angiogenic profile of childhood primitive neuroectodermal brain tumors/medulloblastomas. Eur J Cancer 2001; 37:2064-2072. 17. Hunter SB, Moreno CS. Expression microarray analysis of brain tumors: What have we learned so far. Front Biosci 2002; 7:74-82. 18. Dunn IF, Heese O, McL.Black P. Growth factors in glioma angiogenesis: FGFS, PDGF, EGF, and TGFs. J Neuro-oncol 2000; 50:121-137. 19. Nishie A, Ono M, Shono T et al. Macrophage infiltration and heme oxygenase-1 expression correlate with angiogenesis in human gliomas. Clin Cancer Res 1999; 5:1107-1113. 20. Kazuno M, Tokunaga T, Oshika Y et al. Thrombospondin-2 (TSP2) expression is inversely correlated with vascularity in glioma. Eur J Cancer 1999; 35:502-506. 21. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86:353-364. 22. Machein MR, Kullmer J, Fiebich BL et al. Vascular endothelial growth factor expression, vascular volume, and capillary permeability in human brain tumors. Neurosurgery 1999; 44:732-740. 23. Shweiki D, Itin A, Soffer et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359:843-845. 24. Varlet P, Guillamo JS, Nataf F et al. Vascular endothelial growth factor expression in oligodendrogliomas: A correlative study with Saint-Anne malignancy grade, growth fraction and patient survival. Neuropathol Appl Neurobiol 2000; 4:379-389. 25. Machein MR, Plate KH. VEGF in brain tumors. J Neurooncol 2000; 50:109-120. 26. Johansson M, Brannstrom T, Bergenheim AT et al. Spatial expression of VEGF-A in human glioma. J Neurooncol 2002; 59:1-6. 27. Plate KH, Breir G, Weich HA et al. Vascular endothelial growth factor and glioma angiogenesis: Coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer 1994; 59:520-529.

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28. Hatva E, Kaipainen A, Mentula P et al. Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors. Am J Pathol 1995; 146:368-378. 29. Carroll RS, Zhang J, Bello L et al. KDR activation in astrocytic neoplasms. Cancer 1999; 86:1335-1341. 30. Barleon B, Sozzani S, Zhou et al. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor, flt-1. Blood 1996; 87:3336-3343. 31. Yao Y, Kubota T, Sato K et al. Macrophage infiltration-associated thymidine phosphorylase expression correlates with increased microvessel density and poor prognosis in astrocytic tumors. Clin Cancer Res 2001; 7:4021-4026. 32. Zagzag D, Hooper A, Friedlander DR et al. In situ expression of angiopoietins in astrocytomas identifies angiopoietin-2 as an early marker of tumor angiogenesis. Exp Neuro 1999; 159:391-400. 33. Holash J, Maisonpierre PC, Compton D et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 1999; 284:1994-1998. 34. Hermanson M, Funa K, Hartman M et al. Platelet-derived growth factor and its receptors in human glioma tissue: Expression of mRNA and protein suggests the presence of autocrine and paracrine loops. Cancer Res 1992; 52:3212-3219. 35. Morrison RS, Yamaguchi F, Saya H et al. Basic fibroblast growth factor and fibroblast growth factor receptor 1 are implicated in the growth of human astrocytomas. J Neuro-Oncol 1994; 18:207-216. 36. Wong AJ, Bigner SH, Bigner DD et al. Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci USA 1987; 84:6899-6903. 37. Friedlander M, Brooks PC, Shaffer RW et al. Definition of two angiogenic pathways by distinct alphav integrins. Science 1995; 270:1500-1502. 38. Gladson CL. Expression of integrin alpha v beta 3 in small blood vessels of glioblastoma tumors. J Neuropathol Exp Neurol 1996; 55:1143-1149. 39. Vincenti V, Cassano C, Rocchi M et al. Assignment of the vascular endothelial growth factor gene to the human chromosome 6p21.3. Circulation 1996; 93:1493-1495. 40. Forsythe JA, Jiang BH, Iyer NV et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996; 16:4604-4613. 41. Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996; 271:2746-2753. 42. Fukumura D, Xu L, Chen Y et al. Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo. Cancer Res 2001; 61:6020-6024. 43. Tsai JC, Goldman CK, Gillespie GY. Vascular endothelial growth factor in human glioma cell lines: Induced secretion by egf, pdgf-bb, and bfgf. J Neurosurg 1995; 82:864-873. 44. Maity A, Pore N, Lee J et al. Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3'-kinase and distinct from that induced by hypoxia. Cancer Res 2000; 60:5879-5886. 45. Mukhopadhyay D, Tsiokas L, Sukhatme VP. Wild-type p53 and v-src exert opposing influences on human vascular endothelial growth factor gene expression. Cancer Res 1995; 55:6161-6165. 46. Rak J, Kerbel RS. Ras regulation of vascular endothelial growth factor and angiogenesis. Methods Enzymol 2001; 333:267-283. 47. Feldkamp MM, Lau N, Rak J et al. Normoxic and hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells is mediated by ras. Int J Cancer 1999; 81:118-124. 48. Harada H, Nakagawa K, Iwata S et al. Restoration of wild-type p16 down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human gliomas. Cancer Res 1999; 59:3783-3789. 49. Krieg M, Marti HH, Plate KH. Coexpression of erythropoietin and vascular endothelial growth factor in nervous system tumors associated with von Hippel-Lindau tumor suppressor gene loss of function. Blood 1998; 92:3388-3393. 50. Gorski DH, Beckett MA, Jaskowiak NT et al. Blockade of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res 1999; 59:3374-3378. 51. Park JS, Qiao L, Zao-Zong S et al. Ionizing radiation modulates vascular endothelial growth factor (VEGF) expression through multiple mitogen activated protein kinase dependent pathways. Oncogene 2001; 20:3266-3280. 52. Plate KH, Breir G, Millauer B et al. Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res 1993; 53:5822-5827. 53. Ikeda E, Achen MG, Breier G et al. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells. J Biol Chem 1995; 270:19761-19766. 54. Peoch M, Farion R, Hiou A et al. Immunohistochemical study of VEGF, angiopoietin 2 and their receptors in the neovascularization following microinjection of C6 glioma cells into rat brain. Anticancer Res 2002; 22:2147-2151.

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55. Benjamin LE, Keshet E. Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal. Proc Natl Acad Sci USA 1997; 94:8761-8766. 56. Ke LD, Shi YX, Im SA et al. The relevance of cell proliferation, vascular endothelial growth factor, and basic fibroblast growth factor production to angiogenesis and tumorigenicity in human glioma cell lines. Clin Cancer Res 2000; 6:2562-2572. 57. Ke LD, Shi YX, Yung WK. VEGF (121), VEGF (165) overexpression enhances tumorigenicity in U251MG but not in NG-1 glioma cells. Cancer Res 2002; 62:1854-1861. 58. Cheng SY, Su Huang H-J, Nagane M et al. Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor. Proc Natl Acad Sci USA 1996; 93:8502-8507. 59. Saleh M, Stacker SA, Wilks AF. Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res 1996; 56:393-401. 60. Im SA, Gomez-Manzano C, Fueyo J et al. Antiangiogenesis treatment for gliomas: Transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo. Cancer Res 1999; 59:895-900. 61. Millauer B, Shawver LK, Plate KH et al. Glioblastoma growth inhibited in vivo by a dominant-negative flk-1 mutant. Nature 1994; 367:576-579. 62. Kim KJ, Li B, Winer J et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993; 362:841-844. 63. Rubenstein JL, Kim J, Ozawa T et al. Anti-VEGF antibody reatment of glioblastoma prolongs survival but results in increased vascular cooption. Neoplasia 2000; 2:306-314. 64. Prewitt M, Huber J, Li Y et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res 1999; 59:5209-5218. 65. Stefanik DF, Fellows WK, Rizkalla LR et al. Monoclonal antibodies to vascular endothelial growth factor (VEGF) and the VEGF receptor, flt-1, inhibit the growth of C6 glioma in a mouse xenograft. J Neurooncol 2001; 55:91-100. 66. Wild R, Dhanabal M, Olson TA et al. Inhibition of angiogenesis and tumor growth by VEGF121-toxin conjugate: differential effect on proliferating endothelial cells. Br J Cancer 2000; 83:1077-1083. 67. Holash J, Davis S, Papadopoulos N et al. VEGF-Trap: A VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 2002; 99:11393-11398. 68. Laird AD, Vajkoczy P, Shawver LK et al. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res 2000; 60: 4152-4160. 69. Vajkoczy P, Thurnher A, Hirth KP et al. Measuring VEGF-flk-1 activity and consequences of VEGF-flk-1 targeting in vivo using intravital microscopy: Clinical applications. The Oncologist 2000; 5:16-19. 70. Yuan F, Chen Y, Dellian M et al. Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody. Proc Natl Acad Sci USA 1996; 93:14765-14770. 71. Levin VA, Leibel SA, Gutin PH. Neoplasms of the central nervous system. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer, Principles and Practice of Oncology. 5th ed. Philadelphia: Lippincott-Raven Publishers, 1997:2022-2082. 72. Leibel SA, Scott CB, Loeffler JS. Contemporary approaches to the treatment of malignant gliomas with radiation therapy. Semin Oncol 1994; 21:198-219. 73. Nagashima G, Suzuki R, Asai J et al. Immunohistochemical analysis of reactive astrocytes around glioblastoma: An immunohistochemical study of postmortem glioblastoma cases. Clin Neurol Neurosurg 2002; 104:125-131. 74. Taghian A, Suit H, Pardo F et al. In vitro intrinsic radiation sensitivity of glioblastoma multiforme. Int J Radiat Oncol Biol Phys 1992; 23:55-62. 75. Geng L, Donnelly E, McMahon G et al. Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy. Cancer Res 2001; 61:2413-2419. 76. Chang-Geol L, Heijn M, di Tomaso E et al. Anti-vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. Cancer Res 2000; 60:5565-5570. 77. Kozin SV, Boucher Y, Hicklin DJ et al. Vascular endothelial growth factor receptor-2 blocking antibody potentiates radiation-induced long-term control of human tumor xenografts. Cancer Res 2001; 61:39-44. 78. Senger DR, Galli SJ, Dvorak AM et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983; 219:983-985.

CHAPTER 10

VEGF in Hematopoietic Malignancy Philip T. Murphy and John Quinn

Abstract

I

ncreasing evidence suggests that angiogenesis plays an important role in the pathogenesis of all major hematopoietic malignancies. For example, increased angiogenesis has been correlated with risk of progression in chronic lymphocytic leukemia and lymphomas and with poor prognosis in myeloma and myeloid metaplasia with myelofibrosis. The balance of various positive and negative angiogenic molecules determines the degree of angiogenesis in the bone marrow and elsewhere for hematopoietic cancers, with the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway identified as the most important of the known angiogenic promoters. As well as stimulating new vessel formation to promote tumor growth by conveying oxygen and metabolites, VEGF may stimulate malignant hematopoietic cell growth by paracrine and/or autocrine pathways. The use of anti-angiogenic agents, especially those which target the VEGF/VEGFR pathway, in hematopoietic malignancies appears to be an attractive therapeutic option. Newer compounds such as thalidomide analogues, farnesyl transferase inhibitors and receptor tyrosine kinase inhibitors can target the VEGF-VEGFR pathway more specifically and more potently with a low side effect profile. In the future, these agents may be most effective in combination with other modalities of treatment or as maintenance therapy in hematopoietic cancers.

Introduction Angiogenesis is the process of new blood vessel formation in response to local stimuli. It has been shown to play a major role in solid tumor growth and metastatic spread.1 Small microscopic tumors without vascularization proliferate at a rate equivalent to rapidly growing tumors but have an increased rate of apoptosis. When angiogenesis occurs, proliferation rate of tumor cells remains high but apoptotic rate decreases. Angiogenesis appears to be as important or more so in hematopoietic malignancies, possibly because of the common mesodermal origin of hematopoietic and endothelial cells (EC),2 and, like solid tumors, hematopoietic malignancies appear to progress together with an induction of angiogenesis. Post-natally, the bone marrow (BM) is the principal residence of hematopoietic stem cells and thus the site of origin of the vast majority of hematopoietic malignancies. New blood vessel formation in the BM or increased bone marrow angiogenesis (BMA) appears to play an integral role in the pathophysiology of both myelo- and lymphoproliferative malignancies, including acute lymphoblastic leukemia (ALL),3 acute myeloid leukemia (AML),4,5 myelodysplastic syndromes (MDS),6 chronic lymphocytic leukemia (CLL),7 chronic myeloid leukemia (CML),8 lymphoma,9 multiple myeloma (MM),10,11 and myelofibrosis with myeloid metaplasia (MMM).12 In general, increased angiogenesis is associated with a poor outcome in hematopoietic malignancies6,12-15 and its evaluation may add prognostic information to the predictive value of current staging systems.

VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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Cellular expression of the angiogenic factor VEGF and/or other angiogenic peptides appear to play an important role in the pathophysiology of hematopoietic malignancies, not only as promoters of new vessel formation but also in hematopoietic malignant cell growth and survival by paracrine and/or autocrine mechanisms. Reports of increased VEGF levels in AML, MDS, ALL, CML, chronic myeloproliferative disorders (MPD), CLL, non Hodgkins lymphoma (NHL) and MM support a role for angiogenic factors in the pathophysiology of hematopoietic malignancies and circulating peripheral blood (PB) VEGF levels may be of prognostic value in some hematopoietic cancers. The good preclinical results from blocking VEGF or its receptors in some hematological disorders further indicate VEGF as a leading mediator of angiogenesis.16 Currently, much work is being directed at control of angiogenesis as a therapeutic approach in hematopoietic malignancies.

The VEGF/VEGF Receptor Pathway VEGF appears to be the most endothelial specific and relevant of the known endogenous stimulators of angiogenesis,17 although other molecules may act to promote angiogenesis and tumor cells may synthesize more than one angiogenic factor. In addition, many native inhibitors of angiogenesis have been described. Angiogenesis is thus regulated by a balance of various positive and negative angiogenic molecules.18 There are 5 different VEGF subgroups (VEGF A-E) with VEGF-A (henceforth referred to as VEGF) the most potent angiogenic factor.19 VEGF exerts its effects via high affinity VEGF receptors (VEGFRs), consisting of 3 tyrosine kinase receptors: VEGFR-1 (Flt-1),20 VEGFR-2 (KDR/Flk-1)21 and VEGFR-3 (Flt-4).22 VEGFR-1 and -2 are expressed mainly in vascular EC, whilst VEGFR-3 is predominantly expressed in the lymphatic endothelium. VEGF binds VEGFR-1 and -2 but not VEGFR-3. A fourth receptor, neuropilin-1 (NP-1) binds specifically the 165 amino acid VEGF isoform,VEGF165, and enhances its binding to VEGFR-2, suggesting that it functions as a coreceptor.23 VEGF-C, in conjunction with VEGF-D, regulates lymphangiogenesis by activating VEGFR-3. However, VEGF as well as VEGF-C and VEGF-D promote the proliferation of the target cells primarily through VEGFR-2.24 VEGFR-1 is also expressed on monocytes/macrophages25,26 committed CD34+ hematopoietic progenitor cells27,28 megakaryocytes (MK) and platelets27 whilst VEGFR-2 is expressed on primitive hematopoietic precursors,29 MK and platelets.27,30

Hematopoietic and Endothelial Cells Share a Common Hematopoietic/Endothelial Progenitor Cell During embryogenesis, common hematopoietic/endothelial (HE) precursors share expression of CD34 and VEGFR-2. Post-natally, HE progenitors persist with bilineage differentiation capacity and contribute to long-term hematopoiesis and to the generation of circulating EC, which contribute to the maintenance of endothelium in adults.2,31 These primitive hemangioblast progenitors make up only 0.1-0.5% of total CD34+ cells and have surface phenotype CD34+, CD133+, VEGFR-2+, PECAM+, CXCR-4+, whilst the remaining CD34+, VEGFR-2 negative cells are lineage committed human progenitor cells.29 Under appropriate culture conditions, hematopoietic committment of these precursors is shown by acquisition of CD45 and β1 integrins, whereas angioblasts display the endothelial cell specific marker VE-cadherin (CD144) (Fig. 1). VEGF appears to contribute to the mobilization of hematopoietic and vasculogenic cells into the circulation.32 In healthy adults, most circulating endothelial cells (CEC) derive from vessel wall turnover whereas recent evidence suggests that, in cancer patients, CEC may be recruited from BM-derived CEC progenitors to the peripheral circulation and contribute to angiogenesis associated with tumor growth.33,34

Expression and Function of VEGF in The Normal BM The normal BM is composed of hematopoietic cells and the BM microenvironment, consisting of extracellular matrix (ECM) proteins and BM stromal cells (such as macrophages,

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Figure 1. Surface phenotype of a hemangioblast and its differentiation into either hematopoietic cell or endothelial cell lineages.

fibroblasts, fat cells, osteogenic cells and EC).35 The BM microenvironment plays a critical role in regulating growth and development of hematopoietic cells and is influenced by many complex interactions such as cell-to-cell contacts and secretion of and response to soluble growth factors and cytokines. In the normal BM, MK are the major constitutive producers of VEGF,36 although there is probably also constitutive production by other hematopoietic cells30,37 especially erythroblasts38 and, possibly, macrophages and other stromal cells37,39 but less frequently by myeloid cells.40 Although expression of both VEGFR-1 and VEGFR-2 has been reported in normal BM cells, predominantly myeloid and monocytic cells,30 others have failed to detect VEGFR-2 expression in normal BM or in enriched CD34+ cells, with VEGFR-1 expression only found in a minority of normal BM and CD34+ cell enriched samples.41 VEGF suppresses apoptotic death of normal hematopoietic stem cells without any apparant mitogenic activity or colony-stimulating activity in hematopoietic cells,27 although VEGF may promote the effects of additional colony stimulating factors (CSF) on myeloid colony growth41-43 and via its effects on fibroblasts and EC in normal BM, such as production of granulocyte monocyte-CSF (GM-CSF), interleukin-6 (IL-6) and other cytokines.44 Other biological effects include extracellular matrix (ECM) remodelling and local generation of inflammatory cytokines.29,45

VEGF Bone Marrow Interactions in Hematological Malignancy The importance of EC in the regulation of normal hematopoiesis, including close contact with hematopoietic cells to promote CD34+ cell growth and expansion of human progenitor cells in concert with hematopoietic growth factors,46 further points to an important role for EC in malignant hematopoiesis.

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Figure 2. Possible paracrine and autocrine interactions involving VEGF in the development of hematopoietic malignancy in the bone marrow.

In addition to stimulating production of new blood vessels, VEGF may be involved in complex autocrine and paracrine interactions in the BM microenvironment to promote the development of hematological malignancy (Fig. 2). VEGF may contribute to the pathogenesis of hematological malignancies in one or more of the following ways.

VEGF Stimulates Neoangiogenesis

Malignant human hematopoietic cells secrete VEGF protein,28 which stimulates VEGFR positive EC, resulting in neoangiogenesis and tumor growth by conveying oxygen and metabolites.1 The level of BMA in hematological malignancy is a complex process related to the interaction of various angiogenic and anti-angiogenic factors released into the microenvironment and of cell populations of tumor and host origin with angiogenic and anti-angiogenic activities.47 VEGF also activates EC to become prothrombotic, with increased tissue factor (TF) expression on EC membranes,48,49 resulting in generation of thrombin activity from prothrombin and causing platelet adhesion and activation with platelet release of VEGF.50 The pro-angiogenic effects of TF, thrombin and fibrin may thus be mediated by local adhesion and activation of platelets. Thrombin also activates cancer cells to secrete VEGF with mutual stimulation between EC and cancer cells. VEGF also induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular EC to facilitate EC migration.49

Paracrine Stimulation of Malignant Hematopoietic Cells by VEGFR+ive Cells in the BM Microenvironment Secretion of VEGF by cytokine-stimulated normal hematopoietic progenitor cells may support proliferation of both endothelium and progenitors in a paracrine loop.51 Similarly, VEGF secreting malignant hematopoietic cells may stimulate VEGFR competent cells (EC, other

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stromal cells and/or normal hematopoietic cells) to secrete growth factors for the tumor cells.1 This paracrine interaction would result in a positive feedback that may enhance both angiogenesis and tumor cell survival and self-renewal. It has been proposed that leukemic cell proliferation, angiogenesis and VEGF expression in AML may be linked and contribute to growth advantage of AML via paracrine production of growth factors produced by surrounding EC.42 In fact, EC have been shown to increase proliferation of AML blasts and progenitors and to prevent apoptosis during short term liquid culture, without requiring cell-cell adhesion.52 Resting EC are known to generate stem cell factor (SCF)53 and Flt-3 ligand,54 whilst VEGF-stimulated EC secrete several hematopoietic growth factors, including GM-CSF, granulocyte-CSF (G-CSF), IL-6, SCF and monocyte-CSF (M-CSF), with known stimulatory effects in hematopoietic malignancies.30,41,55,56 IL-6, a major growth and survival factor for myeloma cells, is secreted by BM stromal cells and enhances production and secretion of VEGF by myeloma cells, whilst VEGF secreted by myeloma cells enhances IL-6 production by BM stromal cells, suggesting a paracrine role for VEGF in tumor-stromal interactions in MM.30,57

Stimulation of VEGFR+ive Malignant Cells by VEGF mRNA and/or protein expression for one or more VEGF receptors has been detected in some, but not all, hematopoietic malignant cell lines as well as in fresh samples from patients with various hematopoietic cancers.24,27,28,30,40-42,58-71 In most studies, VEGFR-1 was more readily expressed than VEGFR-2. The functionality of VEGFRs on leukemic cells has also been demonstrated.41,59 As these malignant cells may also produce VEGF, which can mediate proliferation, survival and/or migration of malignant hemopoietic cells,27,28,40,59,66 there is potential for a VEGF/ VEGFR autocrine loop to support angiogenesis independent growth and survival of subsets of hematopoietic malignant cells. The existence of such autocrine stimulation of human hematopoietic malignancy has been suggested in AML and MDS40,59,72,73 and also in adult T cell leukemia (ATL)62 and in primary effusion lymphoma.74 MK are the major constitutive producers of VEGF in the normal BM36 and express both VEGFR-1 and –2.27 In addition, MK are a major source of VEGF in acute megakaryoblastic leukemia (AML-M7)75 whilst elevated expression and secretion of VEGF from human malignant MK cell line CHRF has been shown.39 This suggests that autocrine stimulation of malignant MK by VEGF may also be important in MPD, in particular essential thrombocythemia (ET) and MMM. The severe coagulopathies often found in acute promyelocytic leukemia (APL) could also be partially explained by VEGF acting in an autocrine manner to increase TF expression, an important mediator of coagulation abnormalities in APL, on the leukemic cells.76 In myeloma, however, VEGFR-2 has only been identified on the sorted myeloma cells of one patient57 with either no or only weak expression of VEGFR-1 on MM cell lines and primary cells.30,57 This suggests either that autocrine stimulation is unlikely or that additional biologically active VEGF receptors or mutant forms of VEGFR-1/-2 are expressed on MM cells. In addition,VEGF secreted by stromal cells39 and/or by non malignant hematopoietic cells, may contribute to angiogenesis and/or paracrine stimulation of VEGFR positive hematopoietic malignant cells. In leukemia, EC expressed VEGFR-2 but did not stain for VEGF whilst stromal cells showed a reverse staining pattern.59 In conditions such as MMM, chronic myelomonocytic leukemia (CMML) and fibrosis subsets of MDS, the role of activated fibroblasts in secreting VEGF and other angiogenic factors may be of particular importance. Increased BM microvessel density (MVD) (the average number of microvessels in a microscopic field) in CMML and fibrosis subsets of MDS would, thus, be consistent with reports of VEGF generation by solid tumor-associated macrophages77 and fibroblasts.78,79 In MM, VEGF is produced by both MM cells and BM stromal cells30,57 and adhesion of myeloma cells to BM stromal cells causes increased secretion of VEGF by the latter.80

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Other Interactions of VEGF to Promote Hemopoietic Malignancy

A major source of stored angiogenic factors, including VEGF,81 is the ECM where they can be mobilized by proteases (collagenases), including matrix metalloproteinase-9 (MMP-9), secreted by tumor cells. On the other hand, VEGF has been reported to induce MMP-9 production in certain hematological malignancies.59,82 VEGF and TNFα synergistically increase TF expression in EC,83 leading to increased angiogenesis. VEGF may induce EC to produce basic fibroblast growth factor (bFGF), which may then control angiogenesis as a secondary autocrine or intracrine cytokine,84 whilst the combination of bFGF and VEGF leads to a synergistic effect on EC proliferation.42,85 bFGF may facilitate VEGF-EC interaction through the modulation of EC integrin or VEGFR expression.86,87 VEGF can inhibit maturation of dendritic cells into antigen presenting cells88 and, thus, may help tumors avoid induction of an immune response. In the following sections, the clinical and experimental evidence for the role of VEGF in the pathogenesis of various hematological malignancies will be discussed.

VEGF and Angiogenesis in Acute Leukemia and Myelodysplasia Increased Bone Marrow Angiogenesis Progression of leukemias may be associated with increases in BM EC mass. Intratumoral MVD is the best recognized marker of the degree of angiogenesis.89 Measurement of BM MVD in BM trephine sections may be measured by immunohistochemistry using antibodies (Ab) to CD31, CD34 or Factor VIII-related antigen.90 Since increased angiogenesis in hematological malignancies was first detected, there have been many reports of increased MVD in acute leukemias and MDS compared to controls.3-6,8,42,58,76,91-93 Some studies suggest that increased MVD in AML and MDS may be proportional to percentage of myeloblasts6,4,93 and to expression of VEGF4,42,58,93 and VEGFR-258 in the BM but not to BM cellularity.8 MDS MVD was higher than controls but lower than AML.6 MVD was also higher in the more advanced subsets of MDS, suggesting a correlation between angiogenesis and progression to leukemia in MDS.6 However, MVD has not yet been shown to be an independent prognostic factor in either AML5 or ALL.91 In some reports, MVD reduced towards normal following successful response to therapy.5,42,76,91,93,94

Acute Leukemia Culture Studies and Angiogenesis Although it was recently reported that conditioned medium (CM) from primary AML samples did not increase EC stimulation,92 others have shown increased EC migration and/or proliferation in vitro using AML culture supernatants.41,42 In addition, Rimsza et al noted that AML BM CM from half of patients with unfavorable prognosis supported EC proliferation compared to none of AML BM CM from patients with favourable or intermediate prognosis.95 CM from APL cell line NB4, containing high levels of VEGF, caused strong stimulation of EC migration. The addition of all trans retinoic acid (ATRA) to the CM suppressed VEGF production and inhibited EC migration, thus suggesting that angiogenesis in APL is mediated by VEGF.76 CM from VEGF-producing lymphoblastic leukemic cell lines have also been shown to stimulate proliferation in cultured EC as well as angiogenesis in the murine Matrigel sponge model and the chick embyro chorioallantoic membrane (CAM) assay.96

VEGF mRNA and/or Protein Expression in Acute Leukemia and MDS Most human leukemic cell lines as well as primary isolated leukemias have the capacity to produce VEGF.

Human Leukemic Cell Lines VEGF mRNA and protein is expressed in almost all established human leukemic cell lines.27,28,30,41,59,63,76,96 A strong correlation has been described between the amount of VEGF

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produced in vitro by cultured human leukemia and lymphoma cell lines and both the efficiency of tumor engraftment and the degree of angiogenesis induced by these cells in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Inactivation of VEGF in the culture medium using a Flt-1/Fc chimera inhibited cell proliferation.61

Primary Leukemic Cells VEGF expression and/or secretion has been detected in leukemic cells of ALL, AML and MDS patients,4,40-42,59,63,67,75,76,92,93,97-99 whilst VEGF-C expression was found in 40% of AML samples.24 Myeloblast VEGF protein content has been correlated with shorter survival in AML.97 Others have reported VEGF expression to be an independent poor prognostic factor for overall survival in AML patient subgroups.41,98 Higher cellular VEGF levels in childhood ALL at diagnosis may also be associated with a reduced survival time.99 VEGF expression in the BM of AML patients may be restricted to certain stages of differentiation and maturation of myeloid cells, with correlation to the French American British (FAB) categorization of AML: in AML M0 (AML with minimal evidence of myeloid differentiation), there was a much lower percentage of VEGF expressing blasts compared to all other AML subtypes, where the vast majority of blasts expressed VEGF.75 Lee et al found no correlation between MVD or BM VEGF levels and other hematological or clinical parameters in AML, suggesting that angiogenesis is an independent process.93 In contrast, Litwin et al detected neither increased VEGF secretion nor increased angiogenic potential from CM of cultured AML cells compared to controls and suggested that the increased MVD and increased VEGF immunoreactivity in the BM of AML patients might be a response to microenvironmental factors in vivo rather than an intrinsic property of the leukemic cells.92 Others have reported VEGF expression to be low or below detection limit in MDS and AML blasts, suggesting that MDS or leukemic blasts might not produce clinically relevant VEGF.6 In MDS, VEGF is expressed cytologically by immature myeloid elements, especially leukemic monocytoid precursors.40 BM cellular VEGF protein levels have been found to be higher in MDS than in AML, with increasing VEGF cellular levels associated with shorter survival in MDS. A significant correlation between VEGF and VEGFR-2 levels was also noted for both AML and MDS but without significant correlation between VEGFR levels and duration of survival.67 Only downregulation of VEGFR-2 mRNA inhibited growth of VEGFR-1 and VEGFR-2 expressing HEL cells.28

VEGF Promotes Leukemic Cell Proliferation/Survival/Mobilization Human Cell Lines VEGF caused direct trophic effects on VEGFR positive AML cell lines with increased colony size, number and self-renewal.40 In contrast, VEGF was not mitogenic for CMK 86 human leukemic cell line but reduced apoptosis due to gamma irradiation.27 VEGF reduced apoptosis in HL-60 leukemic cells by induction of Bcl-2 expression.73 VEGFR-2 was constitutively phosphorylated in HEL cells, possibly through autocrine VEGF stimulation, and in cell proliferation experiments, no increase in HEL cell numbers occurred with addition of exogenous VEGF. It is possible that leukemic cells express differing amounts of VEGF or its receptors, which determine whether receptors are constitutively activated or can respond to exogenous VEGF.63 Dias et al describe a novel paracrine angiogenic loop in which VEGF-C, which may be released in increased amounts by EC in response to leukemia derived cytokines, interacts with its receptor VEGFR-3 on leukemic cells lines and primary leukemia cells, to promote leukemia cell survival and proliferation.64 Others support autocrine and/or paracrine stimulation of VEGFR-3 positive leukemic cells by VEGF-C.24

Primary Leukemic Cells

VEGF stimulation of leukemic cell proliferation in vitro28,59,100 may only occur in a subset of AML blasts and may be dependent on the presence of other growth factors (SCF, Flt-3

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ligand, GM-CSF).101 In MDS, Ab neutralization of VEGF inhibited leukemia progenitor formation from most patients with CMML + refractory anemia with excess blasts in transformation (RAEB-t), a subset of MDS, whilst VEGF stimulated leukemia colony formation.40 Others were unable to demonstrate stimulation of AML-colony forming units (CFUs) with VEGF alone,41,42 perhaps due to the fact that leukemic cells express VEGFRs in only a limited number of AML cases28,41,42 or that VEGF may exert other, non proliferative effects on VEGFR positive leukemic cells.

Animal Models

VEGF promoted leukemia growth in vivo.63 The murine leukemic cell line M1 expresses VEGF, VEGFR-2 and NP-1 and produces a chloroma model or systemic leukemia model when injected subcutaneously or intravenously, respectively, into severe combined immunodeficient (SCID) mice. Delivery of VEGF in either model resulted in enhanced tumor growth and vascularization, which was inhibited by VEGF antagonist, soluble NP-1 (sNP-1). As the M1 cell line does not directly respond to VEGF, VEGF and sNP-1 appear to effect angiogenesis and/or paracrine stimulation of the leukemic cells by VEGFR positive EC or other stromal cells.63

Peripheral Blood VEGF Levels Both AML and MDS are associated with increased blood levels of various angiogenic factors, including VEGF.102 Most of these factors appear to be secreted by the neoplastic hematopoietic cells.103 Plasma levels of VEGF, bFGF and hepatocyte growth factor (HGF) were significantly increased in AML and MDS, whilst plasma HGF, TNFα and bFGF but not VEGF were significantly increased in ALL.8 This heterogeneity of factor levels in different disorders suggests that angiogenic factors may play different roles in various leukemias, reflecting the complex nature of interactions between angiogenic factors and BM stroma. In an ongoing prospective study of AML, there was no direct correlation between levels of any one of the above angiogenic factors and BMA.8 This suggests that other factors in the BM stroma or leukemic process may be important in determining the level of vascularity in these diseases or that multiple angiogenic factors may be responsible for overall vascularity. Increased plasma VEGF levels were identified in both untreated AML and MDS patients but increased plasma VEGF was associated with reduced survival in AML but not in MDS.104

VEGF and Angiogenesis in Myeloproliferative Disorders Increased Bone Marrow Angiogenesis

Increased bone marrow angiogenesis has been described in all MPD,105 including systemic mastocytosis.106 BMA was increased in acute and chronic leukemias with maximum MVD in CML, whilst a lack of correlation between BM cellularity and vascularity suggested that vascularity in hematological malignancies is an active and controlled process.8 BM MVD was increased in 70% of MMM patients (compared to 30% and 12% respectively in polycythemia vera (PV) and ET) and correlated with size of spleen, advanced disease and survival, suggesting a role for BMA as a prognostic indicator as well as in the development of MMM.12 In MMM, clonally expanded MK and platelets may have increased VEGF expression (inducible by either paracrine or autocrine mechanisms30,36) as well as elevated release of other angiogenic cytokines transforming growth factor (TGF), bFGF and platelet derived growth factor (PDGF). This suggests that increased BMA may be an integral component of the BM stromal reaction in MMM, in addition to stimulation of fibroblasts and osteoblasts.107 Increased numbers of VEGF positive BM cells correlated with BMA in CML and MMM.108 In contrast, Mesa et al have reported that, despite increased BM MVD in all MMM patients, VEGF immunohistochemical staining of both erythroid precursors and MK was similar or markedly diminished compared to normal BM with corresponding decreases in both urinary VEGF and bFGF. This

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suggests that increased angiogenesis may be independent of VEGF and bFGF and decreases in these cytokines may reflect down-regulation in response to increases in other pro-angiogenic cytokines.109 In MMM, distribution and intensity of BM immunohistochemical staining for VEGF and VEGFR-1 + -2 were not increased but there was microvascular upregulation of TGF-beta receptor I and over-expression of bFGF by MK.110 In ET, BMA was increased (although not universal and less prominent than in MMM or postthrombocythemic myeloid metaplasia (PTMM)) and associated with progressive disease features such as splenomegaly and reticulin fibrosis and marked BM MK proliferation. However, at current follow-up, BMA had no prognostic value for clinical course, vascular events or survival in ET, making it debatable whether increased angiogenesis was merely an epiphenomenon or an actual direct cause of disease progression.111 Cyclooxygenase 2 (COX-2) expression has been found in the BM of chronic phase CML patients with higher levels associated with shorter survival. As COX-2 induces angiogenic factors, including VEGF, increased COX-2 levels may contribute to the increased BM cellularity and VEGF in CML. 112 Matrigel containing murine FL5 12 cells transfected with CML-associated oncogene bcr-abl or bone marrow mononuclear cells (BMMNC) from CML patients, implanted in SCID or BALB/C mice, were significantly more vascularized than implants of nontransfected cells or control BMMNC.82

VEGF mRNA and/or Protein Expression in MPD

Prominent MK VEGF expression has been shown in chronic myeloid disorders.6 The bone marrow of newly diagnosed MPD patients produces increased VEGF but not bFGF in culture medium.113 Increased VEGF mRNA has been detected in BMMNC, PBMNC and PB purified CD34+ cells of MPD patients.114 In systemic mastocytosis, VEGF protein was expressed in areas of mast cell infiltrate in the BM.106 Pretreatment cellular VEGF protein levels in BM samples of chronic phase CML patients are increased and correlate with shorter survival.115 The growth factor-dependent Ba/F3 hematopoietic precursor cell line, when induced to express bcr-abl, produced substantial amounts of VEGF and primary leukemic cells from 2 patients with untreated chronic-phase CML expressed VEGF mRNA constitutively.116 Murine FL5 12 cells transfected with bcr-abl secreted increased amounts of VEGF, whilst myeloblasts derived from BM CD34+ cells of CML patients secreted increased amounts of VEGF, bFGF, HGF and IL-8.82 The bcr-abl targeting drug imatinib mesylate reduced VEGF expression in bcr-abl-positive K562 cells. 117 Bcr-abl-induced VEGF expression may, therefore, contribute to increased angiogenesis in CML and anti-angiogenesis may be part of the anti-CML properties of imatinib mesylate. Increased VEGFR-1 and VEGFR-2 levels in CML BM samples did not correlate with disease phase or other disease features, although increased VEGFR-2 correlated with shortened survival.118

Peripheral Blood VEGF Levels

Increased serum levels of VEGF are consistently reported for all MPD.119 However, in these conditions, clonal MK and platelets contain and/or release increased amounts of VEGF,120 thrombocytosis is frequent and there is evidence of increased platelet activation leading to increased platelet VEGF release. These factors may limit interpretation of serum VEGF, as during the clotting process and separation of the serum, VEGF is released from platelets and WBC.121 To overcome some of these limitations, some studies report plasma VEGF levels or relate serum VEGF to platelet count (VEGF/106 platelets). In fact, both serum VEGF/106 platelets122 and plasma VEGF levels8,123 were significantly increased in MPD. In MMM, serum VEGF levels were approximately 10 times that of controls, although no correlation was found between VEGF and any of the clinical and laboratory features of MMM.120 In MPD and, in particular, in MMM, there are high numbers of circulating CD34+ hematopoietic precursor cells in PB with higher numbers associated with significantly shorter overall survival and time to blast transformation.124 As pluripotent hematopoietic stem cells express VEGFR,29

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it is possible that increased circulating VEGF could influence the excessive production and/or migration of BM clonogenic cells in MPD and especially MMM. Serum VEGF and VEGF/platelet were increased in PV, especially in patients with features of more active disease and in those who had not received chemotherapy.125 As others also found higher serum VEGF levels in MMM patients not receiving chemotherapy,120 it is possible that inhibition of the malignant clone by chemotherapy in MPD may also suppress angiogenesis. In another study of PV patients, those with thrombosis had significantly increased serum VEGF than those without, suggesting an interrelationship between thrombosis and angiogenesis in PV and/or the possibility that increased serum VEGF may reflect a state of platelet activation.126 Others have also found higher plasma VEGF levels in MPD patients with vascular complications,123 implicating VEGF in the endothelial activation noted in MPD patients with thromboembolic complications.127 Increased plasma levels of VEGF, bFGF, HGF and tumor necrosis factor (TNF) α have also been reported in CML,8 whilst a significant correlation between plasma VEGF and the spleen index in CML has been found.123 CML patients on α-interferon (IFNα) were found to have lower PB VEGF levels than untreated or hydroxyurea treated patients.119

VEGF and Angiogenesis in B-Cell Chronic Lymphocytic Leukemia Increasing evidence suggests that angiogenesis is involved in the pathogenesis of B-cell CLL, although it is not yet established whether it is essential for B-CLL.8,128

Increased Angiogenesis in CLL

In limited and heterogenous groups of CLL patients, Kini et al7 reported increased BMA, correlating with Binet stage and BM cellularity, whereas Aguayo et al8 found no increased angiogenesis. In early stage CLL, increased MVD in BM correlated with reduced progression free survival and BM expression of VEGF but not with other predictors of tumor mass or disease progression. This suggested an association between risk of progression and BMA in B cell CLL.13 Increased MVD and VEGF protein was also found in infiltrated lymphoreticular tissues.129 Others confirm a significant increase in MVD in lymph nodes infiltrated by CLL.8,30,68

VEGF mRNA and/or Protein Expression in CLL Although Foss et al could not detect any VEGF mRNA in B-CLL cells by in situ hybridization,79 many other reports have detected in vivo and in vitro VEGF expression in B-CLL cells, both circulating and tissue-phase.13,69,128-132 Hypoxic conditions dramatically upregulated VEGF expression by purified B-CLL cells at both mRNA and protein levels.69,129 VEGF expression on tumor cells was higher in cases with progressive CLL.130 In contrast, lower levels of intracellular VEGF in PB CLL cells were associated with shorter survival times in patients with early-stage disease.131 42% of patients tested had CLL cells expressing Bcl-2 protein, which was negatively correlated with intracellular VEGF level as well as positively correlated with serum bFGF,132 whilst a direct association between stage of CLL and level of Bcl-2 in patients’ cells has been reported.133 Therefore, in CLL, a correlation exists between angiogenesis-related factors and apoptosis-related protein expression. In addition, increased levels of VEGFR-2 protein in PB cells from CLL cases correlated with more advanced disease and significantly shorter survival, making VEGFR-2 a possible prognostic factor in CLL.70 The correlation of increased intracellular VEGF expression with better outcome131 may indicate that VEGFR-2 is an essential factor stimulating cell growth in CLL.

Peripheral Blood VEGF Levels

Plasma VEGF was significantly increased in CLL along with bFGF, TNFα and HGF.8 Serum VEGF and bFGF were also increased in CLL, without correlation between serum and intracellular VEGF levels.132 An aberrant increase of circulating VEGF occurred in only 18% of early B-cell CLL cases but higher serum VEGF levels predicted risk of disease-progression, despite lack of correlation with other biological features representative of tumor mass or disease

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progression. This suggests that serum VEGF might have a possible independent prognostic role in CLL.134 In Binet stage A patients, serum VEGF at diagnosis correlated with PB lymphocytosis, BM histology and beta 2 microglobulin (B2MG) and, again, patients with increased serum VEGF had increased risk of disease progression.135 Serum VEGF/106 platelets for CLL patient groups was increased but did not correlate with disease stage.122 The poor prognostic significance of serum VEGF, irrespective of platelet count, was confirmed in early stage CLL, without correlation between BMA and serum VEGF, perhaps due to contributions to the serum VEGF levels from other sources, such as stromal cells, platelets, and other endothelia.13

VEGF and Angiogenesis in Lymphomas Lymphomas are a heterogenous group of lymphoproliferative malignancies, the best characterized of which is Hodgkin’s disease (HD) because of the presence in biopsy material of the characteristic neoplastic Reed-Sternberg (R-S) cell, a large cell with two or more nuclei, each characterized by a single large nucleolus. The remainder are the non Hodgkin’s lymphoma (NHL), which constitute a wide variety of conditions with differing patterns of behavior and response to treatment, making interpretation of studies on MVD and VEGF especially difficult. Nevertheless, angiogenesis and VEGF could play a cardinal role in the development of many types of NHL. The role of angiogenesis in HD remains even less clear.136 However, using cDNA arrays, mRNA expression of approximately 1000 selected genes were analysed in 21 classical HD tissue samples. All poor outcome HD were characterized by upregulation of genes involved in angiogenesis.137

Increased Angiogenesis in Lymphoma In NHL, increased angiogenesis was associated with tumor progression with significantly higher MVD in high-grade compared to low-grade NHL9,138,139 and also associated with macrophage and mast cell numbers.139,140 However, despite the fact that increased MVD in tumor-involved lymph nodes correlates with disease severity and stage of disease in NHL, no association has been made between angiogenesis and prognosis.9 In the cutaneous T cell NHL, mycosis fungoides, MVD was also increased and correlated with progression.141 In NOD-SCID mice with minimal residual Daudi NHL cell burden, injected human CD34+ cells from patients undergoing PB stem cell mobilization homed to the lymphoma cells, differentiated into EC and increased tumor growth.142 In HD, the extent of neovascularization did not correlate with more advanced disease, whilst MMP-9 expression showed a tendency to poor outcome and correlated with decreased new vessel formation. This suggests that neoplastic new vessel formation is not critical in the progression of HD.143

Lymphoma Culture Studies and Angiogenesis CM from human Burkitt’s lymphoma cell lines stimulate proliferation and/or chemotaxis in cultured EC as well as angiogenesis in different experimental models.96 CM from primary cultures of small lymphocytic lymphoma (SLL) cells also increased EC proliferation.144 bFGF from the malignant cells appeared to be the primary mediator of increased angiogenesis whereas VEGF was not significantly secreted, although culture conditions used an aerobic environment only.144 NHL cells engrafted onto the chick embryo CAM also induced angiogenesis.145

VEGF mRNA and/or Protein Expression in Lymphomas Human Cell Lines

A number of NHL cell lines produced VEGF, although none expressed VEGFR-2 mRNA.30 Burkitt’s lymphoma cell lines produced bFGF and MMPs as well as VEGF.96 Primary effusion lymphoma-derived cell lines expressed both VEGF and VEGFR-1 with intact VEGF induced signalling, suggesting autocrine as well as paracrine effects of VEGF.74 Secretion of VEGF by cell subsets of R-S cell lines has been detected in culture in response to hypoxia.146

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Primary Samples

Although cellular expression of VEGF is common in NHL45,147 and associated with higher tumor grading and high-grade transformation of low-grade NHL, receptor coexpression appears limited to intermediate- or high-grade histology.71 Lymphoma biopsies revealed significant VEGF expression in HD and peripheral T-cell lymphomas but minimal or absent VEGF expression in follicle cell lymphoma and SLL. In lymphoma biopsies, VEGF was mainly expressed by reactive fibroblasts rather than the tumor cells. It was concluded that VEGF may be involved in induction of angiogenesis in peripheral T-cell lymphomas and HD but not low grade B-cell lymphomas.79 In a majority of HD, R-S cells expressed VEGF whilst VEGF was also present in reactive macrophages, ECM and occasionally reactive lymphocytes, without any correlation to subtype of HD or MVD.146 VEGF-C expression is associated with lymphatic vessel development and a study that detected low levels of VEGF-C mRNA in all lymphoma tissues examined suggests that different VEGF homologues may be important in different diseases.148

Peripheral Blood VEGF Levels In a heterogenous group of NHL patients, high pretreatment serum VEGF was associated with poor outcome. As higher serum VEGF was also more likely in large cell NHL and associated with high serum lactate dehydrogenase (LDH), serum VEGF levels appeared to be related to growth of NHL and active angiogenesis.149 Simultaneous elevations of VEGF and bFGF were associated with poor survival in different grades of lymphomas and in the largest subgroup, the large-cell diffuse and immunoblastic lymphomas, with serum VEGF an independent prognostic factor irrespective of platelet and white cell counts.150 Both serum VEGF and IL-6 may be independent prognostic factors for overall survival of aggressive lymphoma151 whilst elevated plasma VEGF and bFGF levels correlated with reduced event free survival in NHL.152

VEGF and Angiogenesis in Myeloma Angiogenesis appears to have an integral role in MM progression and correlates with prognosis and resistance to chemotherapy.

Increased Bone Marrow Angiogenesis

In addition to reports of increased BMA in myeloma patients,153,154 other studies show a correlation between increased MVD in BM of myeloma patients and disease progression and poor prognosis.10,11,14,15,155-159 Increased BMA has also been reported in an animal model of human MM.160 MM is the only hematological malignancy in which the prognostic relevance of BMA has been shown in multivariate analysis.11,14 In contrast, Ahn et al could not demonstrate any consistent association between increased MVD and outcome in myeloma.153 Many studies report a correlation between BMA and plasma cell labelling index (PCLI),10,11,159 suggesting proliferating myeloma cell secretion of angiogenic cytokines such as VEGF,30 although MVD may also have an adverse effect on survival independent of PCLI.157 A significant correlation between BM MVD, myeloma cell proliferation and BM VEGF positivity has been shown.161 BMA has also been positively correlated with tumor burden15,153,155 and some studies have shown a reduction in BM MVD following a partial or complete response to chemotherapy. Although a reduction in BMA to normal values has been reported,162 BM MVD tends to remain elevated even after successful chemotherapy, suggesting residual disease. Others have found no significant reduction in BMA after standard chemotherapy157 or even after high dose chemotherapy with autologous peripheral stem cell rescue.156 BM MVD in MM patients is increased compared to monoclonal gammopathy of undetermined significance (MGUS)10 and increased BMA may be implicated in the mechanisms of progression from MGUS or nonactive MM to active MM.155 In patients with solitary bone plasmacytoma, a localized plasma cell (PC) malignancy that may progress to myeloma, BMA was low in all cases but patients with high-grade angiogenesis in their plasmacytomas were

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more likely to progress to myeloma and to have a shorter progression-free survival, providing further evidence of the importance of angiogenesis in MM pathogenesis.163

VEGF mRNA and/or Protein Expression in Myeloma Both malignant plasma cells and BM stromal cells express and secrete VEGF within the bone marrow of myeloma patients,30,57,161 whereas VEGF is poorly expressed by benign plasma cells from normal bone marrow.36 VEGF overexpression and secretion by MM cell lines has also been demonstrated.30,57

VEGF Stimulates Myeloma Cell Proliferation in Vitro VEGF can directly trigger growth and migration of MM cell line, MM1S, as well as patient MM and plasma cell leukemia (PCL) cells.66 Using MM patient derived material, Ab neutralization of VEGF inhibited in vitro colony growth, whilst VEGF stimulated colony formation.30

Peripheral Blood VEGF Levels Serum levels of VEGF and HGF in MM patients were elevated and significantly higher in patients with poor response to chemotherapy. VEGF levels were also higher in patients with extramedullary plasmacytomas, suggesting a contributory role for VEGF in extramedullary expansion of tumor cells. However, serum VEGF levels did not correlate with disease severity of MM, as indicated by disease stage and B2MG levels.164 Successful therapy of MM was associated with decrease in serum levels of VEGF, bFGF and HGF, but without an association between increased serum VEGF levels and stage of disease.165 Because of a lack of correlation between serum VEGF levels and MM severity or tumor angiogenesis, serum VEGF levels can not be regarded as a surrogate marker of angiogenesis in MM.166 VEGF, bFGF, and HGF levels were higher in plasma from BM aspirate than from PB in MM patients, suggesting the BM environment as their major source, whilst VEGF correlated with features of disease activity such as c-reactive protein and B2MG. This suggests that several soluble factors may play a role in the angiogenic activity in MM but their contribution to myeloma progression may be different.167

VEGF Signalling Pathways As a Therapeutic Target in Hematological Malignancies In the preceding sections, evidence has been presented for increased angiogenesis in the pathogenesis of hematological malignancies. The VEGF/VEGFR angiogenic pathway appears to be the most important contributor to increased angiogenesis in the majority of these malignancies as well as stimulating survival, growth and migration of some subsets of malignant hematopoietic cells by paracrine and/or autocrine mechanisms. At the present time, much effort is being directed at developing potential therapeutic interventions to block the effect of VEGF on the vasculature in hematopoietic malignancies in an attempt to selectively retard the growth of malignant cells and increase their susceptibility to apoptotic signals from the microenvironment and ultimately, to improve prognosis. Strategies that target VEGF and its receptors may have limited toxicity as limited, if any, damage would be expected on normal nondividing EC. Despite this, other toxicities could occur, possibly due to the role of VEGF in maintaining vascular integrity. A further drawback to the use of small molecules that target the function of VEGFR or Ab to VEGF and VEGFRs is the need for prolonged and frequent administration of high doses. However, although generally still in phase I, II or III clinical trials, studies of both Ab to VEGF and blockers of the VEGFR tyrosine kinase pathway indicate that these agents are safe and may have clinical efficacy in hematological malignancies. Angiogenesis inhibitors are mainly cytostatic agents that target dividing EC, which are genetically stable, homogenous and have a low mutation rate and are less likely to develop drug-resistant mutations.168 However, targeting a single, pro-angiogenic molecule might

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ultimately result in resistance to therapy as tumor cells increase production of other pro-angiogenic molecules. Therefore, to overcome classical drug resistance and improve patient survival, agents that target VEGF and its receptors may have optimal effect in combination with conventional therapies and/or other anti-angiogenic and biological agents to target both the microenvironment and tumor compartments. In addition, some hematological malignancies may now be approached as chronic but treatable disease with the potential of using VEGF/VEGFR pathway inhibitors as single agents after standard treatment, when tumor burden is low, to induce further tumor regression or as maintenance treatment for ‘tumor stabilization’.169 Such single-agent therapy may also be appropriate in patients with disseminated but less rapidly growing tumors such as early MDS or smouldering MM to delay or prevent disease progression. It is possible that different anti-angiogenic drugs may be targeted to specific stages of the cancer to be most effective.170 Thalidomide can induce marked and durable clinical response in myeloma, even in patients refractory to conventional or high dose therapy.171-173 In addition to inhibition of VEGF-induced BMA, thalidomide may also block VEGF-induced direct effects on MM cell growth, survival and migration.174 However, the lack of change in BM MVD in myeloma patients responding to thalidomide suggests other anti-myeloma mechanisms may be more important,174 although others report reduction in plasma and/or BM VEGF and bFGF in thalidomide responders.175,176 A decrease in CEC in myeloma patients responding to thalidomide also supports an anti-angiogenic mechanism.177 Thalidomide has also demonstrated therapeutic activity in MDS178 and MPD, especially MMM.119,179,180 Immunomodulatory drugs (ImiDs) are thalidomide analogues with stronger anti-angiogenic activity, which reduce VEGF secretion.181 In a SCID model of human MM, tumor growth and associated angiogenesis was inhibited and host survival prolonged to a greater extent with ImiDs than with thalidomide.182 ImiDs have produced clinical improvement in refractory or relapsed MM.183 Other agents which can reduce VEGF secretion in MM include arsenic trioxide184 and 2-methoxyestradiol, a natural metabolite of estradiol with potent anti-angiogenic effects.185 IFNα confers its anti-tumor activity partly by its anti-angiogenic activity, resulting from inhibition of VEGF transcription.186 Patients with CML treated with IFNα had reduction in BM MVD.105 CML patients on IFNα had VEGF and HGF levels lower than untreated or hydroxyurea treated cases, suggesting a possible anti-angiogenic mechanism of IFNα.119 Other agents with anti-VEGF effects include ATRA,76 linomide,187 pentoxyfylline,188 imatinib mesylate117 and carboxyamidotriazole.189 COX-2 induces the expression of angiogenic factors, including VEGF, and is therefore a potential target for an anti-angiogenic approach in hematological malignancies.190 Selective COX-2 inhibitors can significantly reduce VEGF expression in preclinical models,191 whilst aspirin inhibits EC tube formation by CM from human cancer cell lines via suppression of VEGF-induced COX-2 expression.192 SCH66336 and R115777 are farnesyl transferase inhibitors (FTI), which inhibit ras protooncogene activation, an upstream regulator of VEGF, essential for EC proliferation. R115777 in accelerated/blastic CML, MMM and MM showed some some clinical response in a minority of CML and MMM cases. Responding patients had higher pre treatment plasma VEGF which decreased significantly during therapy.193 Neutralizing VEGF Ab have shown leukemic growth inhibition in vitro40 and completely inhibited angiogenesis induced by CM from the APL cell line, NB4.76 An anti-VEGF mAb inhibited growth of human cancer cell lines injected into nude mice but had no effect on the growth rate of tumor cells in vitro.16 Bevacizumab is a humanized anti-VEGF Ab which produces sustained neutralization of circulating VEGF and has shown potent anti-tumor activity in experimental models.194 The use of soluble VEGFRs can be used to compete with the wild-type full-length receptor for VEGF. Expression of the soluble truncated form of the VEGFR-1 receptor, sFlt-1, by gene therapy inhibited growth of human cancer cells in nude mice195 whilst VEGF inactivation

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from the culture medium of human leukemic and lymphoma cell lines by a Flt-1/Fc chimera inhibited cell proliferation.61 In a systemic murine leukemia model, survival of mice injected with adenovirus encoding for sNP-1 dimer (the soluble version of the extracellular portion of NP-1 which can bind circulating VEGF and displace tumor cell NP-1-bound VEGF) was prolonged with reduction in leukemia burden and reduced BM neovascularization.63 Other possible means of inhibiting the VEGF-VEGFR pathway might include antisense strategies or ribozymes to decrease VEGF production.60 VEGF as well as VEGF-C and VEGF-D promote the proliferation of target cells primarily through VEGFR-222 and VEGF-C is expressed in 40% of AML samples.24 Therefore, inhibition of VEGRFR-2 signalling may provide an effective means of blocking both the paracrine and autocrine signalling induced by various forms of VEGFs in hematopoietic malignancies.72 Tyrosine kinase inhibitors of VEGFRs interfere with proliferation and function of stromal cells in long-term cultures from normal BM with reduced levels of GM-CSF and IL-6.44 This suggests that VEGFR inhibitors may also help in neoplastic BM diseases, including stromal cell diseases such as MMM, by reducing stimulatory signals from the microenvironment. Receptor tyrosine kinase inhibitors (RTKI) against ligand induced autophosphorylation of VEGFR-1 and VEGFR-2 include SU5416,196 SU6668 and PTK787.197 SU6668 also inhibits bFGFR and PDGFR whilst SU5416 inhibits c-kit. As c-kit, whose ligand is SCF, is expressed by >10% of myeloblasts in up to 80% of AML cases, a combination of angiogenesis inhibition with c-kit inhibition may be an especially useful approach in AML therapy.198 SU5416 can inhibit the clonogenic response to VEGF in the human leukemia cell line KG1.199 Single agent SU5416 has inhibited leukemic growth in vivo94 and has had modest clinical activity in a phase II study of refractory AML or MDS200 and minimal clinical activity in refractory bcr-abl negative MPD.201 PTK787 directly inhibits proliferation of MM cell lines and primary MM cells which express VEGFR-1. It also blocks VEGF-induced migration of MM cells and inhibits the increased MM cell proliferation and increased IL-6 and VEGF secretion in cultures of MM cells adherent to BM stromal cells.65 For VEGFR-2 positive human leukemic cell lines, blockade of VEGF binding to its receptor by a chimeric anti-VEGFR-2 neutralizing Ab, IMC-1C11, resulted in reduced cell survival and reduced VEGF-induced leukemic cell migration in vitro. This mAb also inhibited VEGF-induced proliferation and migration of human leukemic cells in vitro and prolonged survival of NOD-SCID mice inoculated with human primary leukemia and leukemic cell lines, in association with decrease in plasma human VEGF levels.59 Two fully human anti-VEGFR-2 Ab have shown even higher affinity binding to VEGFR-2 and greater inhibition of leukemia growth in the NOD-SCID mouse model.202 There was also a direct correlation between antigen-binding affinity of the Ab and their ability to inhibit VEGF stimulation of human leukemia cells in vitro and to prolong survival of the mice. An oral DNA vaccine against VEGFR-2 has proven to be effective immunotherapy as it treated pre-existing malignancy and prevented formation of cancer in mice. The vaccine evoked a durable T cell-mediated immune response against EC at sites of active angiogenesis, followed by collapse of tumor vasculature, without effects on normal, quiescent vasculature.203 Other approaches to block VEGFR-2 have included dominant-negative receptor mutants and germ-line disruption of VEGFR genes.204

Conclusion Evidence has been presented that increased angiogenesis has a major role in the development and progression of hematopoietic malignancies. Although it has been argued that increased angiogenesis could merely be an epiphenomenon or a surrogate marker of other more important biological features of these conditions, clinical studies and in vitro experiments suggest that angiogenesis is an integral part of their pathogenesis. In particular, increased angiogenesis has been correlated with poor prognosis in MM11 and in MMM12 and with risk of progression in B-cell CLL13 and in NHL139 and there is evidence of a reduction in angiogenesis

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following successful response to therapy in acute leukemia5,94 and in MM.162 In addition, there is increased BMA in AML compared to MDS6 and in MM compared to MGUS,10 implicating angiogenesis in the mechanism of progression. Although angiogenesis is regulated by a balance of many possible pro- and anti-angiogenic molecules, the VEGF-VEGFR angiogenic pathway appears to be the most important promoter of angiogenesis in hematopoietic malignancies. The importance of this pathway may be partly due to the common VEGFR-2 positive hematopoietic/endothelial (HE) precursor with bilineage differentiation capacity into either hematopoietic cells or EC. VEGF may not only stimulate VEGFR positive cells to induce angiogenesis, but may also result in hematopoietic malignant cell stimulation by paracrine and/or autocrine means. Based on the apparent importance of angiogenesis in the pathogenesis of hematopoietic malignancies, the use of anti-angiogenic agents and, in particular, compounds to interfere with the VEGF/VEGFR pathway may be an attractive option to improve treatment of these disorders. Already, agents, such as thalidomide171 and IFNα,119 whose modes of action may include some degree of anti-angiogenesis, have shown clinical efficacy. The more recent development of newer, more potent compounds designed to more specifically target the VEGF/VEGFR system, whilst having low side effect profiles, offers great promise. Such compounds as ImiDs,183 FTIs193 and RTKIs200 have already shown variable clinical efficacy in initial trials when used as single agent therapy in patients resistant to conventional therapies. However, it is likely that anti-VEGF/VEGFR agents will be more useful in combination with conventional therapies and/or other modalities as first line treatment. Such agents could also find a place as maintenance therapy in chronic hematological malignancies with minimal residual disease or in early MDS/smouldering MM to reduce progression to more active malignancy. In these latter situations, a number of anti-angiogenic agents might need to be used together to reduce the risk of resistance.

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138. Vacca A, Ribatti D, Roncali L et al. Dammacco F. Angiogenesis in B cell lymphoproliferative diseases. Biological and clinical studies Leuk Lymphoma 1995; 20:27-38. 139. Vacca A, Ribatti D, Ruco L et al. Angiogenesis extent and macrophage density increase simultaneously with pathological progression in B-cell non hodgkin’s lymphoma. Br J Cancer 1999; 79:965-970. 140. Ribatti D, Nico B, Vacca A et al. Do mast cells help to induce angiogenesis in B-cell non hodgkin’s lymphomas? Br J Cancer 1998; 77:1900-1906. 141. Vacca A, Moretti S, Ribatti D et al. Progression of mycosis fungoides is associated with changes in angiogenesis and expression of the matrix metalloproteinases 2 and 9. Eur J Cancer 1997; 33:1685-1692. 142. de Bont ES, Guikema JE, Scherpen F et al. Mobilized human CD34+ hematopoietic stem cells enhance tumor growth in a nonobese diabetic/severe combined immunodeficient mouse model of human non hodgkin’s lymphoma. Cancer Res 2001; 61:7654-7659. 143. Kuittinen O, Soini Y, Turpeenniemi-Hujanen T. Diverse role of MMP-2 and MMP-9 in the clinicopathological behavior of Hodgkin’s lymphoma. Eur J Haematol 2002; 69:205-212. 144. Rimsza L, Pastos K, Massey K et al. Endothelial stimulation by small lymphocytic lymphoma correlates with secreted levels of basic fibroblast growth factor. Br J Haematol 2003; 120:753-758. 145. Ribatti D, Vacca A, Bertossi M et al. Angiogenesis induced by B-cell non hodgkin’s lymphomas. Lack of correlation with tumor malignancy and immunologic phenotype. Anticancer Res 1990; 10:401-406. 146. Doussis-Anagnostopoulou IA, Talks KL, Turley H et al. Vascular endothelial growth factor (VEGF) is expressed by neoplastic Hodgkin-Reed-Sternberg cells in Hodgkin’s disease. J Pathol 2002; 197:677-683. 147. Dvorak HF, Sioussat TM, Brown LF et al. Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: Concentration in tumor blood vessels. J Exp Med 1991; 174:1275-1278. 148. Salven P, Lymboussaki A, Heikkila P et al. Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in human tumors. Am J Pathol 1998; 153:103-108. 149. Salven P, Teerenhovi L, Joensuu H. A high pretreatment serum vascular endothelial growth factor concentration is associated with poor outcome in non hodgkin’s lymphoma. Blood 1997; 90:3167-3172. 150. Salven P, Orpana A, Teerenhovi L et al. Joensuu H. Simultaneous elevation in the serum concentrations of the angiogenic factors VEGF and bFGF is an independent predictor of poor prognosis in non Hodgkin’s lymphoma: A single-institution study of 200 patients. Blood 2000; 96:3712-3718. 151. Niitsu N, Okamato M, Nakamine H et al. Simultaneous elevation of the serum concentrations of vascular endothelial growth factor and interleukin-6 as independent predictors of prognosis in aggressive non hodgkin’s lymphoma. Eur J Haematol 2002; 68:91-100. 152. Bertolini F, Paolucci M, Peccatori F et al. Angiogenic growth factors and endostatin in non Hodgkin’s lymphoma. Br J Haematol 1999; 106:504-509. 153. Ahn MJ, Park CK, Choi JH et al. Clinical significance of microvessel density in multiple myeloma patients. J Korean Med Sci 2001; 16:45-50. 154. Laroche M, Brousset P, Ludot I et al. Increased vascularization in myeloma. Eur J Haematol 2001; 66:89-93. 155. Vacca A, Ribatti D, Presta M et al. Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma. Blood 1999; 93:3064-3073. 156. Rajkumar SV, Fonseca R, Witzig TE et al. Bone marrow angiogenesis in patients achieving complete response after stem cell transplantation for multiple myeloma. Leukemia 1999; 13:469-472. 157. Kumar S, Fonseca R, Dispenzieri A et al. Bone marrow angiogenesis in multiple myeloma: Effect of therapy. Br J Haematol 2002; 119:665-671. 158. Niemoller K, Jakob C, Heider U et al. Bone marrow angiogenesis and its correlation with other disease characteristics in multiple myeloma in stage I versus stage II-III. J Cancer Res Clin Oncol 2003; 129:234-238. 159. Munshi N, Wilson CS, Penn J et al. Angiogenesis in newly diagnosed multiple myeloma: Poor prognosis with increased microvessel density (MVD) in bone marrow biopsies. Blood 1998; 92:98a. 160. Yaccoby S, Barlogie B, Epstein J. Primary myeloma cells growing in SCID-hu mice: A model for studying the biology and treatment of myeloma and its manifestations. Blood 1998; 92:2908-2913. 161. Xu JL, Lai R, Kinoshita T et al. Proliferation, apoptosis and intratumoral vascularity in multiple myeloma: Correlation with clinical stage and cytological grade. J Clin Pathol 2002; 55:530-534. 162. Sezer O, Niemoller K, Kaufmann O et al. Decrease in bone marrow angiogenesis in myeloma patients achieving a remission after chemotherapy. Eur J Haematol 2001; 66:238-244.

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163. Kumar S, Fonseca R, Dispenzieri A et al. Prognostic value of angiogenesis in solitary bone plasmacytoma. Blood 2003; 101:1715-1717. 164. Iwasaki T, Hamano T, Ogata A et al. Clinical significance of vascular endothelial growth factor and hepatocyte growth factor in multiple myeloma. Br J Haematol 2002; 116:796-802. 165. Sezer O, Jakob C, Eucker J et al. Serum levels of the angiogenic cytokines basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) multiple myeloma. Eur J Haematol 2001; 66:83-88. 166. Jakob C, Zavrski I, Heider U et al. Serum levels of VEGF do not correlate with the severity of multiple myeloma. Br J Haematol 2002; 119:276. 167. Di Ramondo F, Azzaro MP, Palumbo G et al. Angiogenic factors in multiple myeloma: Higher levels in bone marrow than in peripheral blood. Haematologica 2000; 85:800-805. 168. Boehm T, Folkman J, Browder T et al. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 1997; 390:404-407. 169. Harris AL. Antiangiogenesis for cancer therapy. Lancet 1997; 349:13-15. 170. Talks KL, Harris AL. Current status of anti-angiogenic factors. Br J Haematol 2000; 109:477-489. 171. Singhal S, Hehta J, Desikan R et al. Anti-tumor activity of thalidomide in refractory multiple myeloma. N Engl J Med 1999; 341:1566-1571. 172. Barlogie B, Desikan R, Eddlemon P et al. Extended survival in advanced and refractory multiple myeloma after single-agent thalidomide: Identification of prognostic factors in a phase 2 study of 169 patients. Blood 2001; 98:492-494. 173. Tosi P, Ronconi S, Zamagni E et al. Salvage therapy with thalidomide in multiple myeloma patients relapsing after autologous peripheral blood stem cell transplantation. Haematologica 2001; 86:409-413. 174. Raje N, Anderson K. Thalidomide – a revival story. N Engl J Med 1999; 341:1606-1609. 175. Dmoszynska A, Bojarska-Junak A, Domanski D et al. Production of pro-angiogenic cytokines during thalidomide treatment of multiple myeloma. Leuk Lymphoma 2002; 43:401-406. 176. Pitini V, Teti D, Arrigo C et al. Thalidomide treatment of relapsed multiple myeloma patients and changes in circulating VEGF and bFGF. Br J Haematol 2002; 119:275. 177. Zhao J, Zhang H, Smith ELP et al. Circulating endothelial cells are prognostic in multiple myeloma. Blood 2002; 100:394a. 178. Strupp C, Germing U, Aivado M et al. Thalidomide for the treatment of patients with myelodysplastic syndromes. Leukemia 2002; 16:1-6. 179. Elliott MA, Mesa RA, Chin-Yang L et al. Thalidomide therapy in myelofibrosis with myeloid metaplasia. Br J Haematol 2002; 117:288-296. 180. Piccaluga PP, Visani G, Pileri SA et al. Clinical efficacy and anti-angiogenic activity of thalidomide in myelofibrosis with myeloid metaplasia. A pilot study Leukemia 2002; 16:1609-1614. 181. Dredge K, Marriott JB, Macdonald CD et al. Novel thalidomide analogues display anti-angiogenic activity independently of immunomodulatory effects. Br J Cancer 2002; 87:1166-1172. 182. Lentzsch S, Rogers MS, LeBlanc R et al. S-3-Amino-phthalimido-glutarimide inhibits angiogenesis and growth of B-cell neoplasias in mice. Cancer Res 2002; 62:2300-2305. 183. Richardson P, Schlossman RL, Weller E et al. Immunomodulatory drug CC-5013 overcomes drug resistance and is well tolerated in patients with relapsed multiple myeloma. Blood 2002; 100:3063-3067. 184. Hayashi T, Hideshima T, Akiyama M et al. Arsenic trioxide inhibits growth of human multiple myeloma cells in the bone marrow microenvironment. Mol Cancer Therapeut 2002; 1:851-860. 185. Chauhan D, Catley L, Hideshima T et al. 2-methoxyestradiol (2ME) overcomes drug resistance in multiple myeloma cells. Blood 2002; 100:2187-2194. 186. Von Marschall Z, Scholz A, Cramer T et al. Effects of interferon α on vascular endothelial growth factor gene transcription and tumor angiogenesis. J Natl Cancer Inst 2003; 95:437-448. 187. Gross DJ, Reibstein I, Weiss L et al. The anti-angiogenic agent linomide inhibits the growth rate of von Hippel-Lindau paraganglioma xenografts to mice. Clin Cancer Res 1999; 5:3669-3675. 188. Amirkhosravi A, Meyer T, Warnes G et al. Pentoxyfylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor. Thromb Haemost 1998; 80:598-602. 189. Waselenko JK, Shinn CA, Willis CR et al. Carboxyamido-triazole (CAI)—a novel “static” signal transduction inhibitor induces apoptosis in human B-cell chronic lymphocytic leukemia cells. Leuk Lymphoma 2001; 42:1049-1053. 190. Fosslien E. Molecular pathology of cyclooxygenase-2 in neoplasia. Ann Clin Lab Sci 2000; 30:3-21. 191. Williams CS, Tsujii M, Reese J et al. Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest 2000; 105:1589-1594.

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192. Shtivelband MI, Juneja H, Lee S et al. Supression of cancer medium- and VEGF-induced endothelial tube formation by aspirin and sodium salicylate. Blood 2002; 100:682a. 193. Cortes J, Albitar M, Thomas D et al. Efficacy of the farnesyl transferase inhibitor R115777 in chronic myeloid leukemia and other hematologic malignancies. Blood 2003; 101:1692-1697. 194. Presta LG, Chen H, O’Connor SJ et al. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997; 57:4593-4599. 195. Goldman CK, Kendall RL, Cabrere G et al. Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate. Proc Natl Acad Sci USA 1998; 95:8795-8800. 196. O’Farrell AM. Analysis of mechanism of action and biomarkers for kinase inhibitor SU5416 in acute myeloid leukemia patients. J Clin Oncol 2002; 21:70a. 197. Wood JM, Bold G, Buchdunger E et al. PTK787/ZK222584, a novel and potent inhibitor of vascular endothelial growth factor receptor kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2000; 60:2178-2189. 198. Spiekermann K, Faber F, Voswinckel R et al. The protein tyrosine kinase inhibitor SU5614 inhibits VEGF-induced endothelial cell sprouting and induces growth arrest and apoptosis by inhibition of c-kit in AML cells. Exp Hematol 2002; 30:767-773. 199. Smolich BD, Yuen HA, West KA et al. The anti-angiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts. Blood 2001; 97:1413-1421. 200. Giles FJ, Stopeck AT, Silverman LR et al. SU5416, a small molecule tyrosine kinase receptor inhibitor, biological activity in patients with refractory acute myeloid leukemia or myelodysplastic syndromes. Blood 2003; 102:795-801. 201. Giles FJ, Cooper MA, Silverman L et al. Phase II study of SU5416 - a small-molecule, vascular endothelial growth factor tyrosine-kinase receptor inhibitor – in patients with refractory myeloproliferative diseases. Cancer 2003; 97:1920-1928. 202. Zhu Z, Hattori K, Zhang H et al. Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity. Leukemia 2003; 17:604-611. 203. Niethammer AG, Xiang R, Becker JC et al. A DNA Vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 2002; 8:1369-1375. 204. O’Reilly MS. Vessel maneuvers: Vaccine targets tumor vasculature. Nat Med 2002; 8:1352-1353.

CHAPTER 11

Targeting VEGF in Pancreatic Cancer Cheryl H. Baker, Carmen C. Solorzano and Isaiah J. Fidler

Abstract

T

he progressive growth and metastasis of neoplasms, including pancreatic cancers, depend, in part, on angiogenesis, the extent of which is determined by the balance between pro-angiogenic and anti-angiogenic molecules released by tumor cells and normal host cells. Since vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis, we determined whether blocking VEGF receptor signaling using a novel tyrosine kinase inhibitor (PTK787) decreases the growth and metastasis of human cancers growing orthotopically in nude mice. PTK787 is a potent inhibitor of VEGF tyrosine kinases and has been shown to be active in submicromolar concentrations. Work from our laboratory has recently shown that oral administrations of PTK787 can inhibit vascular permeability in mice and hence prevent formation of malignant ascites associated with ovarian cancer or pleural effusions associated with advanced lung adenocarcinomas. In this chapter, we discuss the investigation of the inhibitory effects of PTK787 on the growth and metastasis of human pancreatic cancer cells implanted into the pancreas of nude mice. We show that PTK787 combined with conventional therapy (gemcitabine) leads to significant therapeutic effects, which are mediated in part by induction of apoptosis in tumor-associated endothelial cells.

Cancer Metastasis The major cause of death from cancer is due to metastases that are resistant to conventional therapy.1,2 Several reasons defined by Paget’s “seed and soil” hypothesis account for the failure to treat metastasis.3 First, tumors are biologically heterogeneous and contain multiple cell populations with different sensitivities to various cytotoxic agents, and with different angiogenic, invasive and metastatic properties.1,2 The metastases can be located in different lymph nodes and different organs and the specific organ environment can influence the biological behavior of the metastatic cells, including their response to therapies.1,2,4,5 Clearly, a better understanding of the molecular mechanisms that regulate the complex interactions between the metastatic cells and host factors will allow for more effective designed therapies and new options for the treatment of cancer patients.

Tumor Angiogenesis Because a tumor mass that exceeds 0.25 mm in diameter can no longer receive oxygen and nutrients by diffusion, the continuous growth of tumors and the development of metastasis require the generation of a vasculature, i.e., angiogenesis.6,8 The process of angiogenesis consists of a series of linked and sequential steps that ultimately lead to the establishment of a new vascular bed (Fig. 1). To generate capillary sprouts, endothelial cells must proliferate, migrate, degrade the basement membrane, and form a structure, i.e., a new lumen organization.9 To stimulate angiogenesis both tumor cells and host cells secrete a variety of factors. At present, more than a dozen pro-angiogenic molecules have been reported, including basic fibroblast VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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Figure 1. The angiogenic cascade. Angiogenesis is a series of linked and sequential steps that ultimately lead to the establishment of a neovascular blood supply to the tumor mass. Angiogenesis begins with an angiogenic stimulus followed by local degradation of the basement membrane surrounding the capillaries. Endothelial cell migration is then accompanied by the proliferation of cells at the leading edge of the migrating column. As they move, the endothelial cells begin to organize into three-dimensional structures to form new capillary tubes. Endothelial cell survival occurs which then allows for the interplay of numerous factors, which can act in a positive or negative fashion. For angiogenesis to occur, this five-step process must take place. Adapted from Baker CH, Sloreano CC, Fidler IJ. Angiogenesis and cancer metastasis: Angiogenic therapy of human pancreatic adenocarcinoma. Int J Clin Oncol 2001; 6:60.

growth factor (bFGF), vascular endothelial growth factor, also known as vascular permeability factor (VEGF/VPF)10,13 interleukin-8 (IL-8), angiogenin, angiotropin, platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factor (PDGF), transforming growth factor-α (TGF-α), epidermal growth factor (EGF), transforming growth factor β (TGF-β) and tumor necrosis factor-α (TNF-α).6,11 Many tissues and tumors, however, also generate factors that inhibit angiogenesis. The angiogenic phenotype of a tissue or a tumor is therefore determined by the net balance between positive and negative regulators of neovascularization.14 The complexity of the angiogenic process suggests the existence of multiple controls of the system that can be temporarily turned on and off.12 This principle of angiogenesis is valid for numerous solid malignant tumors, making anti-angiogenic strategies a unifying concept in cancer therapy. Advantages of anti-angiogenic therapy include (i) broad spectrum of activity, (ii) targeting of a genetically stable cell population (endothelial vs. tumor cells), thereby reducing the chance of drug resistance, (iii) relatively nontoxic, and (iv) possible synergism with other anticancer therapies.15

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Regulation of Angiogenesis by the Microenvironment The production of VEGF, bFGF and IL-8 by tumor cells or host cells or the release of angiogenic molecules from the extracellular matrix induces the growth of endothelial cells and formation of blood vessels. Recent data from our laboratory have demonstrated that the organ environment can directly contribute to the induction and maintenance of the angiogenic factors bFGF16,17 and IL-8.18 For example, in renal cell carcinoma patients, the level of bFGF in the serum or urine inversely correlates with survival.19,20 Human renal cancer cells implanted into different organs of nude mice had different metastatic potentials: those implanted into the kidney produced a high incidence of lung metastasis, whereas those implanted subcutaneously were not metastatic.16 The subcutaneous (or intramuscular) implanted tumors had a lower level of mRNA transcripts for bFGF than did continuously cultured cells, whereas tumors implanted in the kidney of nude mice had 20-fold the levels of bFGF mRNA and protein level than those of cultured cells and ectopic implanted tumors. Histopathological examination of the tumors revealed that subcutaneous tumors had few blood vessels, whereas the tumors in the kidney had many blood vessels.16,17 Constitutive expression of IL-8 directly correlates with the metastatic potential of human melanoma cells.18 IL-8 contributes to angiogenesis by inducing proliferation, migration, and invasion of endothelial cells.21 Several organ-derived cytokines (produced by inflammatory cells) can upregulate expression of IL-8 in normal and tumorigenic cells.22 IL-8 expression was upregulated in coculture of melanoma cells with keratinocytes (skin), whereas it was inhibited in cells cocultured with hepatocytes (liver). Similar results obtained with conditioned media from keratinocyte and hepatocyte cultures suggested that organ-derived factors, e.g., IL-1 and TGF-β, can modulate the expression of IL-8 in human melanoma cells.21 The influence of the microenvironment on the expression of VEGF/VPF, angiogenesis, tumor cell proliferation, and metastasis was investigated using human gastric cancer cells implanted in orthotopic (stomach) and ectopic (subcutaneous) sites in nude mice. Tumors growing in the stomach wall were highly vascularized and expressed higher levels of VEGF/VPF than did subcutaneous tumors.23 Moreover, only tumors implanted in the stomach produced metastasis, suggesting that the expression of VEGF/VPF, vascularization and metastasis of human gastric cancer cells are regulated by the organ microenvironment.23

VEGF/VPF: A Pro-Angiogenic Molecule Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) was initially detected as a factor secreted by tumor cells into tissue culture medium or ascites fluid in vivo.24 The factor was identified as a heparin-binding protein of molecular mass 34-42 kD and was termed VPF. It was later demonstrated that VPF can also stimulate division of endothelial cells.25 Independently, several groups isolated a secreted protein, which they called VEGF because it had selective mitogenic activity for cultured endothelial cells.26,27 On the basis of amino acid and cDNA sequence analysis, it is now known that VEGF and VPF are identical,28 and VEGF is the more commonly used term for this angiogenic factor. VEGF/VPF is a homodimeric heparin-binding glycoprotein that exits in at least four isoforms through alternative splicing of the primary mRNA transcript. The major isoforms are designated VEGF121, VEGF165, VEGF189 and VEGF205 according to the number of amino acids each protein contains.28 The vascular permeability induced by VEGF/VPF is 50,000 times that induced by histamine, the standard for induction of permeability.29 Increased permeability allows for the diffusion of proteins into the interstitium on which endothelial cells migrate. In addition, VEGF plays a major role as a survival factor for dividing and nondividing endothelial cells30-33 and plays an important physiological role in blood vessel formation as demonstrated by the finding that loss of a single VEGF allele results in embryonic lethality.34,35 The VEGF/VPF receptors Flk-1/KDR (Fetal liver kinase 1, the murine homologue of human Kinase insert Domain-containing Receptor) and Flt-1 (fms-like tyrosine kinase) are ex-

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pressed almost exclusively on endothelial cells.28,35 Rarely, expression of the various VEGF/ VPF receptors has been demonstrated on cells of neural origin, Kaposi’s sarcoma cells, hematopoietic precursor cells, and some tumor cell types.36,37 VEGF binds and mediates its activity through kinase receptors Flk-1/KDR and Flt-1. Deletion of the genes encoding Flk-1 and Flt-1 in knockout mice are embryonic lethal.38,39 These receptors are tyrosine kinase receptors that require dimerization in order to induce intracellular signaling.

Regulation of VEGF/VPF Expression in Tumors Tumor cells may constitutively overexpress angiogenic factors or they produce them in response to external stimuli. The most potent external stimulus of angiogenic factor expression is hypoxia40,41 occurring as a consequence of poor perfusion. The hypoxia-induced increase in angiogenic factor expression represents a response by cells trying to survive. Hypoxia increases angiogenic factor expression by inducing signal cascade pathways that eventually lead to an increase in transcription of VEGF/VPF as well as stabilization of the mRNA transcript. Hypoxia induction of VEGF is probably mediated through Src kinase activity, which then leads to downstream induction of signaling cascades and eventually stabilization of hypoxia-inducible factor-Iα (HIF-Iα).42 This factor then increases the transcription of the VEGF/VPF gene, which in turn leads to the induction of angiogenesis.43,46

Development of a Human Pancreatic Adenocarcinoma Model Critical to our studies has been the development of relevant in vivo models for cancer metastasis,47,48 which have produced conclusive evidence that the outcome of metastasis is regulated by the interaction of unique tumor cells with homeostatic mechanisms.1,2 Indeed, studies from our laboratory and others have shown that malignant human tumors implanted into orthotopic organs of immunodeficient mice are highly vascularized, grow progressively, and produce distant metastasis, whereas implantation of the same tumors in ectopic organs does not lead to extensive angiogenesis or production of metastasis.49,57 Two general methods have been used to select metastatic variants in vivo. In the first, tumor cells are implanted into the orthotopic organs, and spontaneous metastases from distant organs are isolated and reinjected into orthotopic organs. In the second method, tumor cells are introduced into the circulation to produce lesions in distant organs, or experimental metastases. Spontaneous metastases occur subsequent to completion of all steps of the metastatic process, whereas experimental metastases reflect only the ability of tumor cells to survive in the circulation, arrest in a distant capillary bed, and grow in a distant organ parenchyma.1,2,4,7,48,58 Depending on the neoplasm, the selection process is gradual, requiring several cycles of in vivo selection.49,54,56 This cyclic procedure was originally used to isolate the B16-F10 metastatic cell line from the wild-type B16 murine melanoma cell line59 and, subsequently, human renal cell carcinoma,50 colon carcinoma,49,50 transitional cell carcinoma,52,53 and prostate carcinoma.55,56 To develop an orthotopic model for metastasis of human pancreatic cancer, our laboratory used the COLO 375 human pancreatic cancer cell line originally established by Morgan et al from a celiac axis lymph node that had been partially replaced by neoplastic foci of well-differentiated, mucin-containing pancreatic ducts.60 Vezeridis et al subsequently injected the fast-growing (FG) variant line of the COLO 375 cells into the spleen of nude mice.61 The in vivo isolated cells designated L3.3 produced liver lesions at a higher incidence than the original COLO 375 cells.61 Bruns et al injected the L3.3 cells into the spleen or the pancreas of nude mice. Experimental liver metastases (spleen injection) or spontaneous liver metastases (pancreas injection) were harvested, established in culture, and designated L3.4sl (spleen-liver) and L3.4pl (pancreas-liver).62 The cultures were harvested and injected into the spleen or pancreas of another set of nude mice. The cycles were repeated twice more to yield lines L3.6sl and L3.6pl. This study led to the development of a reproducible orthotopic model to study the biology of human pancreatic cancer metastasis, with variant lines with enhanced metastatic properties. Moreover, both in vitro cultured cells and in vivo tumors derived from L3.6pl and

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L3.6sl cells expressed higher levels of VEGF/VPF, bFGF, IL-8, and MMP-9 than did the low metastatic FG cells or tumors.62

Anti-VEGF Therapy in Pancreatic Cancer As stated above, the progressive growth and metastasis of neoplasms, including pancreatic cancers, depend in part, on angiogenesis,7 the extent of which is determined by the balance between pro-angiogenic and anti-angiogenic molecules released by tumor cells and normal host cells.6,10,14 Extensive local invasion and early lymphatic and hematogenous metastasis characterize cancer of the exocrine pancreas.63,64 At the time of diagnosis, more than 80% of patients present with either locally advanced or metastatic disease.65 The inability to detect pancreatic cancer at an early stage, its aggressiveness, and the lack of effective systemic therapy are responsible for rapid death from this disease. Only 1-4% of all patients with adenocarcinoma of the pancreas will survive 5 years after diagnosis.66,67 For patients with advanced pancreatic cancer, recent introduction of the deoxycytidine analogue gemcitabine does not extend median survival beyond 6 months.68 The design of more effective therapy is therefore a major goal of pancreatic cancer research. The role of several anti-angiogenic strategies aimed at inhibiting VEGF activity, including neutralizing anti-VEGF antibodies,69,70 anti-VEGF receptor antibodies,71,73 competition by soluble VEGF receptors, antisense VEGF mRNA-expressing constructs74,75 and VEGF receptor tyrosine kinase inhibitors39,76,77 has been recently described. VEGF receptors play a critical role in endothelial cell function, making them ideal targets for treatment of deregulated angiogenesis.7 Indeed, retrovirus-mediated expression of a dominant-negative Flk-1 VEGF-R has been shown to suppress the growth of glioblastoma.78,79 Inhibitors of KDR tyrosine kinases or antibodies to KDR have been shown to inhibit growth of tumors subcutis71,77,80 as well as colon cancer liver metastasis.76 Based on these prior studies we hypothesized that blockade of VEGF-R signaling is an important anti-angiogenic therapeutic modality for inhibiting the growth and preventing the metastasis of human pancreatic cancer. We have investigated the inhibitory effects of a potent inhibitor of VEGF receptor tyrosine kinases on the growth and metastasis of human pancreatic cancer cells implanted into the pancreas of nude mice. We found that daily oral administrations of the VEGF receptor tyrosine kinase inhibitor, PTK787 (75 mg/kg) combined with biweekly intra-peritoneal injections of standard chemotherapy, gemcitabine (125 mg/kg) produced significant therapeutic effects, which were mediated in part by induction of apoptosis in tumor associated endothelial cells. Treatment with the inhibitor alone or gemcitabine alone reduced the growth of primary pancreatic neoplasms by 60% and the combination reduced it by 80%; the combination also reduced the incidence of liver metastasis (5/10 to 1/10). This combination therapy significantly prolonged survival by extending it from 1 month to 4 months. The daily oral administrations of the inhibitor were well tolerated by the mice.81 Immunohistochemistry analyses of the pancreatic tumors demonstrated significant decreases in microvessel density (MVD) and in proliferating tumor cells with an associated increase in apoptotic tumor cells in the inhibitor alone and combination treated groups. Moreover, double staining of tumors with an antibody to CD31 to identify endothelial cells and TUNEL (Terminal deoxynucleotidyltransferase-mediated dUTP Nick End Labeling) to identify apoptotic cells, suggested that the reduction in microvessel density was due to a significant increase in endothelial cell apoptosis (Fig. 2). A significant increase in the number of apoptotic endothelial cells (CD31/TUNEL positive cells in 10 random fields of view) was found in pancreatic tumors harvested 28 days after the initiation of treatment with PTK787 (5 ± 2) or PTK787 plus gemcitabine (9 ± 2) as compared to control tumors (0.1 ± 0.4) or gemcitabine-treated tumors (1.8 ± 1).81 Expression of VEGF and its receptors is associated with increased tumor vascularity and decreased survival in human pancreatic cancer.80,82,83 In this study, the oral administration of the VEGF receptor tyrosine kinase inhibitor did not decrease the expression of VEGF, bFGF

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Figure 2. Immunofluorescent CD31 (endothelial cells), TUNEL (apoptosis) double labeling of human pancreatic cancer growing in the pancreas of nude mice. Pancreatic tumors (L3.6pl cells) from mice treated with HBSS (control), gemcitabine, PTK 787, or gemcitabine plus PTK 787 were harvested on day 28 of the treatment. The tissues were processed for immunohistochemistry using anti-CD31 antibodies (Texas Red) and TUNEL (FITC-green). A representative sample (x400) of this CD31/TUNEL fluorescent double staining is shown. Fluorescent red: CD31-positive endothelial cells; fluorescent green: TUNEL-positive cells; yellow: TUNEL-positive endothelial cells. Adapted from Baker CH, Sloreano CC, Fidler IJ. Angiogenesis and cancer metastasis: Angiogenic therapy of human pancreatic adenocarcinoma. Int J Clin Oncol 2001; 6:62.

or IL-8 in treated neoplasms, but was associated with a significant decrease in microvessel density (MVD), i.e., decreased angiogenesis to the neoplasms. VEGF tyrosine kinase receptors are mostly expressed on endothelial cells, so it is unlikely that treatment with this inhibitor alone would directly lead to apoptosis of tumor cells. Indeed, our results suggest that the increase in tumor cell apoptosis resulted from the preceding death of endothelial cells. These results are supported by recent reports where VEGF is implicated as a survival factor for endothelial cells.30,33 In addition, KDR activation by VEGF leads to KDR upregulation; blocking this positive feedback mechanism (by preventing KDR activation) may further decrease VEGF-R signaling.84 The decrease in VEGF-R signaling may interfere with the survival/recovery of dividing endothelial cells affected by gemcitabine and result in apoptosis in tumor-associated endothelial cells.

Anti-Angiogenic Therapy: Clinical Implications The understanding that angiogenesis is essential for tumor growth and metastasis formation has led to a large effort to discover effective anti-angiogenic compounds including those

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directed against VEGF or its receptors. Since primary tumor growth is often controlled with surgery and/or irradiation, anti-angiogenic agents may be most beneficial to prevent widespread metastatic disease. To succeed, several principles must be considered. First, physiologic angiogenesis is important in reproduction and wound healing as well as physiological responses to cardiac ischaemia or peripheral vascular disease. Thus, a balance must be maintained between the therapeutic and toxic effects, inhibiting angiogenesis in the tumor without preventing physiological angiogenesis crucial to homeostasis. Second, any anti-angiogenic therapy including those directed against VEGF signaling is complicated by its chronic nature since this type of therapy is not cytotoxic but rather only prevents progressive growth of a tumor. Therefore, since anti-angiogenic therapy is designed to inhibit the development of new blood vessels and existing tumor vessels, the end points for success or failure must be redefined. For example, the early endpoint may require measurement of surrogate markers and tumor stabilization, rather than traditional endpoints such as tumor shrinkage. The best clinical results may be obtained by a combination therapy, as observed in treatment of pancreatic carcinoma described above. Regardless, the central role of VEGF in angiogenesis, tumor growth, progression and metastasis provides a promising therapeutic target.

References 1. Fidler IJ. Critical factors in the biology of human cancer metastasis: Twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res 1990; 50:6130-6138. 2. Fidler IJ. Modulation of the organ microenvironment for the treatment of cancer metastasis. J Natl Cancer Inst 1995; 84:1588-1592. 3. Paget S. The distribution of secondary growths in the cancer of the breast. Lancet 1889; 1:571-573. 4. Fidler IJ, Kripke ML. Metastasis results from preexisting variant cells within a malignant tumor. Science 1977; 197:893-895. 5. Fidler IJ, Talmadge JE. Evidence that intravenously derived murine pulmonary melanoma metastases can originate from the expansion of a single tumor cell. Cancer Res 1986; 46:5167-5171. 6. Fidler IJ, Ellis LM. The implication of angiogenesis to the biology and therapy of cancer metastasis. Cell 1994; 47:185-188. 7. Folkman J. Angiogenesis in cancer, vascular, rheumatoid, and other disease. Nature Med 1995; 1:27-31. 8. Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: An imbalance of positive and negative regulation. Cell 1991; 64:327-336. 9. Auerbach W, Auerbach R. Angiogenesis inhibition: A review. Pharmac Ther 1994; 63:265-311. 10. Folkman J, Klagsbrun M. Angiogenic factors. Science 1987; 235:444-447. 11. Bouck N, Stellmach V, Hsu SC. How tumors become angiogenic. Adv Cancer Res 1996; 69:135-174. 12. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333:1757-1763. 13. Cockerill GW, Gamble JR, Vadas MA. Angiogenesis: Models and modulators. Int Rev Cytol 1995; 159:113-160. 14. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996; 86:353-364. 15. Kerbel RS. Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents. Bioessays 1991; 13:31-36. 16. Singh RK, Bucana CD, Gutman M et al. Organ site-dependent expression of basic fibroblast growth factor in human renal cell carcinoma cells. Am J Pathol 1994; 145:365-374. 17. Singh RK, Gutman M, Bucana CD et al. Interferons alpha and beta downregulate the expression of basic fibroblast growth factor in human carcinomas. Proc Natl Acad Sci USA 1995; 92:4562-4566. 18. Singh RK, Gutman M, Radinsky R et al. Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice. Cancer Res 1994; 54:3242-3247. 19. Nanus DM, Schmitz-Drager BJ, Motzer RJ et al. Expression of basic fibroblast growth factor in primary human renal tumors: correlation with poor survival. J Natl Cancer Inst 1994; 85:1587-1599. 20. Nguyen M, Watanabe H, Budson AE et al. Elevated levels of an angiogenic peptide, basic fibroblast growth factor, in the urine of patients with a wide spectrum of cancers. J Natl Cancer Inst 1994; 86:356-361. 21. Leek RD, Harris AL, Lewis CE. Cytokine networks in solid human tumors: regulation of angiogenesis. J Leukoc Biol 1994; 56:423-435.

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22. Gutman M, Singh RK, Bucana CD et al. Regulation of IL-8 expression in human melanoma cells by the organ environment. Cancer Res 1995; 55:2470-2475. 23. Takahashi Y, Mai M, Wilson MR et al. Site-dependent expression of vascular endothelial growth factor, angiogenesis, and proliferation in human gastric carcinoma. Int J Oncol 1996; 8:701-705. 24. Senger DR, Galli SJ, Dvorak AM et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983; 219:983-985. 25. Connolly DT, Heuvelman DM, Nelson R et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989; 84:1470-1478. 26. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989; 161:851-858. 27. Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci USA 1989; 86:7311-7315. 28. Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J Biol Chem 1996; 271:603-606. 29. Dvorak HF, Brown LF, Detmar M. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995; 146:1029-1039. 30. Kumar R, Yoneda J, Bucana CD et al. Regulation of distinct steps of angiogenesis by different angiogenic molecules. Int J Oncol 1998; 12:749-757. 31. Gerber HP, McMurtrey A, Kowalski J et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 1998; 273:30336-30343. 32. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998; 273:13313-13316. 33. Nor JE, Christensen J, Mooney DJ et al. Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl- 2 expression. Am J Pathol 1999; 154:375-384. 34. Ferrara N, Carver-Moore K, Chen H et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996; 380:439-442. 35. Carmeliet P, Ferreira V, Breier G et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380:435-439. 36. Zeigler BL, Valtieri M, Porada GA et al. KDR receptor: A key marker defining hematopoietic stem cells. Science 1999; 285:1553-1558. 37. Ferrer FA, Miller LJ, Lindquist R et al. Expression of vascular endothelial growth factor receptors in human prostate cancer. Urology 1999; 53:567-571. 38. Shalaby F, Rossant J, Yamaguchi TP et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995; 376:62-66. 39. Fong GH, Rossant J, Gertsenstein M et al. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 1995; 376:66-70. 40. Shweiki D, Itin A, Stoffer D et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359:843-845. 41. Levy AP, Levy NS, Wegner S et al. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem 1995; 270:13333-13340. 42. Ellis LM, Staley CA, Liu W et al. Downregulation of vascular endothelial growth factor in human colon carcinoma cell line transfected with an antisense expression vector specific for c-src. J Biol Chem 1998; 273:1052-1057. 43. Marti HJ, Bernaudin M, Bellail A et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol 2000; 156:965-976. 44. Minet E, Michel G, Remacle J et al. Role of HIF-1 as a transcription factor involved in embryonic development, cancer progression and apoptosis. Int J Molec Med 2000; 5:252-258. 45. Kimura H, Weisz A, Kurashima Y et al. Hypoxia response element of the human vascular endothelial growth factor gene mediated transcriptional regulation by nitric oxide: Control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000; 95:189-197. 46. Ryan HE, Lo J, Johnson RS. HIF-1α is required for solid tumor formation and embryonic vascularization. EMBO J 1998; 17:3005-3015. 47. Fidler IJ. Orthotopic implantation of human colon carcinomas into nude mice provides a valuable model of the biology and therapy of cancer metastasis. Cancer Metastasis Rev 1991; 10:229-243. 48. Fidler IJ. Rationale and methods for the use of nude mice to study the biology and therapy of human cancer metastasis. Cancer Metastasis Rev 1986; 29:29-49.

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49. Giavazzi R, Campbell DE, Jessup JM et al. Metastatic behavior of tumor cells isolated from primary and metastatic human colorectal carcinomas implanted into different sites in nude mice. Cancer Res 1986; 46:1928-1933. 50. Morikawa K, Walker SM, Jessup JM et al. In vivo selection of highly metastatic cells from surgical specimens of different primary human colorectal carcinomas implanted into nude mice. Cancer Res 1988; 48:1943-1948. 51. Naito S, von Eschenbach AC, Giavazzi R et al. Growth and metastasis of tumor cells isolated from renal cell carcinoma implanted into different organs of nude mice. Cancer Res 1986; 46:4109-4115. 52. Price JE, Polyzos A, Zhang RD et al. Tumorigenicity and metastasis of human breast carcinoma lines in nude mice. Cancer Res 1990; 50:717-721. 53. Ahlering TE, Dubeau L, Jones PA. A new in vivo model to study invasion and metastasis of human bladder carcinoma. Cancer Res 1985; 47:6660-6665. 54. Dinney CPN, Fishbeck R, Singh RK et al. Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice. J Urol 1995; 154:1532-1538. 55. McLemore TL, Liu MC, Blacker PC et al. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice. Cancer Res 1987; 47:5132-5140. 56. Stephenson RA, Dinney CPN, Gohji K et al. Metastatic model for human prostate cancer using orthotopic implantation in nude mice. J Natl Cancer Inst 1992; 84:951-957. 57. Pettaway CA, Pathak, S, Greene G et al. Selection of highly metastatic variants of different human prostate carcinomas utilizing orthotopic implantation in nude mice. Clin Cancer Res 1996; 2:1627-1636. 58. Aukerman SL, Price JE, Fidler IJ. Different deficiencies in the prevention of tumorigenic-low-metastatic murine K-1735 melanoma cells from producing metastases. J Natl Cancer Inst 1986; 77:915-924. 59. Fidler IJ. Selection of successive tumor lines for metastasis. Nature 1973; 242:148-149. 60. Morgan RT, Woods LK, Moore GE et al. Human cell line (COLO375) of metastatic pancreatic adenocarcinoma. Int J Cancer 1980; 25:1627-1636. 61. Vezeridis MP, Tzanakakis GN, Meitner PA et al. In vivo selection of a highly metastatic cell line of a human pancreatic carcinoma in the nude mouse. Cancer 1992; 69:2060-2063. 62. Bruns CJ, Harbison MT, Kuniyasu H et al. In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia 1999; 1:50-62. 63. Warshaw AL, Fernandez-del CC. Pancreatic carcinoma. N Engl J Med 1992; 326:455-465. 64. Evans DB, Abbruzzese JL, Rich T. Cancer of the Pancreas. In: deVita V, Hellman S, Rosenberg S, eds. Principles and Practice of Oncology Philadelphia: Lippincott JB, 1997:1054-1087. 65. Wanebo HJ, Vezeridis MP. Pancreatic carcinoma in perspective: A continuing challenge. Cancer 1996; 78:580-591. 66. Landis SH, Murray T, Bolden S et al. Cancer Statistics, 1999. CA. Cancer J Clin 1999; 49:8-31. 67. Fernandez E, La Vecchia C, Porta M et al. Trends in pancreatic cancer mortality in Europe, 1955-1989. Int J Cancer 1994; 57:786-792. 68. Burris HA, Moore MJ, Andersen J et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: A randomized trial. J Clin Oncol 1997; 15:2403-2413. 69. Kim KJ, Li B, Winer J et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993; 362:841-844. 70. Asano M, Yukita A, Matsumoto T et al. Inhibition of tumor growth and metastasis by an immunoneutralizing monoclonal antibody to human vascular endothelial growth factor/vascular permeability factor 121. Cancer Res 1995; 55:5296-5301. 71. Prewett M, Huber J, Li Y et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res 1999; 59:5209-5218. 72. Witte L, Hicklin DJ, Zhu Z et al. Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/ KDR) as an anti-angiogenic therapeutic strategy. Cancer Metastasis Rev 1998; 17:155-161. 73. Zhu Z, Rockwell P, Lu D et al. Inhibition of vascular endothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodies from a phage display library. Cancer Res 1998; 58:3209-3214. 74. Neufeld G, Cohen T, Gengrinovitch S et al. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13:9-22. 75. Cheng SY, Huang HJ, Nagane M et al. Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor. Proc Natl Acad Sci USA 1996; 93:8502-8507.

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76. Shaheen RM, Davis DW, Liu W et al. Anti-angiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis. Cancer Res 1999; 59:5412-5416. 77. Fong TA, Shawver LK, Sun L et al. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res 1999; 59:99-106. 78. Millauer B, Shawver LK, Plate KH et al. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 1994; 367:576-579. 79. Millauer B, Longhi MP, Plate KH et al. Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo. Cancer Res 1996; 56:1615-1620. 80. Ikeda N, Adachi M, Taki T et al. Prognostic significance of angiogenesis in human pancreatic cancer. Br J Cancer 1999; 79:553-1563. 81. Solorzano, CC, Baker CH, Bruns CJ et al. Inhibition of growth and metastasis of human pancreatic cancer growing in nude mice by PTK 787/ZK222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases. Cancer Biother and Radiopharm 2001; 359-370. 82. Brown LF, Berse B, Jackman RW et al. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract. Cancer Res 1993; 53:4727-4735. 83. Itakura J, Ishiwata T, Shen B et al. Concomitant over-expression of vascular endothelial growth factor and its receptors in pancreatic cancer. Int J Cancer 2000; 85:27-34. 84. Shen BQ, Lee DY, Gerber HP et al. Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro. J Biol Chem 1998; 273:29979-29985.

CHAPTER 12

Effects of Fibrinogen and Associated Peptide Fragments on the Activation of Human Endothelial Cells by VEGF in Vitro Carolyn A. Staton, Nicola J. Brown and Claire E. Lewis

Abstract

A

ngiogenesis, the development of new blood vessels from existing vasculature involves the migration, proliferation and differentiation of endothelial cells and is crucial for the growth and metastasis of tumors. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a potent cytokine that is central in both normal vasculogenesis in embryonic development and the neovascularization associated with numerous pathologies including cancer. Hemostatic mechanisms regulate blood flow by controlling platelet adhesion and fibrin deposition and hemostatic proteins co-ordinate the spatial localization and temporal sequence of clot/endothelial cell stabilization followed by growth and repair of the damaged blood vessel. A number of hemostatic proteins have been shown to regulate angiogenesis either directly, by interacting with endothelial cells themselves, or indirectly by interacting with other regulators of angiogenesis, including VEGF. The polypeptide, fibrinogen, is the central protein in the hemostasis pathway. Here, we review the evidence for the ability of this protein (and various protein fragments derived from it) to modulate the stimulatory effects of VEGF on human microvascular endothelial cells in vitro.

Introduction Both angiogenesis and hemostasis are usually quiescent but remain poised for tissue repair. Hemostatic proteins ensure that endothelial cells are initially stabilized to allow clot formation to occur, and then trigger the process of endothelial activation and angiogenesis for the repair of the damaged blood vessel.1 The sequence and inter-relationships of these processes are essential. Negative regulators of angiogenesis are initially required to immobilise the endothelial cells for clot anchorage, after which pro-angiogenic factors stimulate endothelial cells to become mobile, and dismantle the cross-linked fibrin structure to rebuild a vessel wall. This suggests that many of the hemostatic proteins are likely to regulate angiogenesis and may therefore influence such angiogenesis-dependent diseases as cancer. This chapter focuses on the central hemostatic protein, fibrinogen (Fgn) and its degradation products, and outlines their effects on VEGF-stimulated angiogenic mechanisms in vitro. During wound healing, Fgn is cleaved to form a temporary matrix meshwork of fibrin fibres, which stabilizes into a blood clot incorporating other factors, including growth factors and enzymes, into the meshwork. This temporary matrix is rapidly broken down, by a process called fibrinolysis, as the vessel wall is repaired. The structure and formation of Fgn and fibrin and the generation of the various degradation products by fibrinolysis are described in the next two VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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sections of the chapter. The effects of Fgn and related fragments on the process of VEGF-induced angiogenesis are then discussed.

Fibrinogen and Fibrin Formation Fgn is a key protein in the hemostatic pathway and is found in two main sites within the body, in the plasma and on platelets. Fgn has a molecular weight of 340 kD and is a dimer consisting of three pairs of non-identical polypeptide chains, termed α, β, and γ chains.2 Both chicken and bovine Fgn have been crystallized and their structures shown to be trinodular3-4 with the polypeptide chains interlinked to give three globular domains, two larger outlying D-domains and a central E-domain, which contains the amino-terminal portions of the six polypeptide chains strongly interconnected by disulphide bridges (Fig.1A). The domains are held together by alpha helices within the structures of the α, β, and γ chains which form coiled-coils. The conversion of Fgn to fibrin involves a number of steps5 (Fig. 1B). The first step involves thrombin cleavage of the α and β chains by hydrolyzing two specific arginine-glycine bonds between residues 16 and 17 on the α chain and residues 14 and 15 on the β chain to release the fibrinopeptides, FpA and FpB, respectively.6 The removal of the fibrinopeptides stimulates electrostatic, non-covalent bonding between the E and D-domains of adjacent fibrin monomers to yield half staggered two-stranded fibrin protofibrils.7 The model proposed by Fowler et al8 assumed that as the protofibrils enlarged, there was an increased association laterally and interactions between D-domains of adjacent fibrin monomers would allow the formation of multi-stranded fibrils under the action of factor XIIIa, which stabilizes the structure by cross-linking the fibrin monomers within both the α and γ chains (Fig. 1B).

Fibrinolysis Fibrinolysis, the degradation of cross-linked fibrin by plasminogen, is of vital importance not only for the removal of temporary fibrin, but also to provide both a safeguard against the clot embolizing in the general circulation, and a mechanism for recanalization of thrombosed vessels. Fibrinolysis occurs in an ordered sequence under the control of plasmin, which is associated with physical changes in the structure of the fibrin clot. Plasmin digestion of the fibrin monomer occurs in the same manner as plasmin digestion of Fgn.9,10 It appears that plasmin digests the fibrin monomer at several lysyl- and arginyl bonds, in a stepwise manner, starting with a symmetrical digest to produce fragment X (240 kD). Within fragment X the α chains are degraded at their carboxy-termini and a small peptide β15-42 cleaved from the amino-termini of the β-chains. Plasmin further degrades fragment X, asymmetrically, to produce one D-fragment (92 kD) and an unstable fragment Y (consisting of a combined D-fragment and E-fragment). The latter is rapidly degraded by plasmin to release a second D-fragment and E-fragment (50 kD) containing the amino terminal core region of Fgn. Thus, the digestion of one fibrin monomer by plasmin, produces two D-fragments and one E-fragment. Within the fibrin polymer, the D-domains from different fibrin monomers are covalently cross-linked between the 2 γ-chains. Thus when cross-linked fibrin polymers are degraded by plasmin, an additional fragment is seen, the cross-linked D-dimer (Fig. 1C).11-13

Effects of Fibrinogen on Endothelial Cell Activation Disturbances in blood coagulation occur in patients with various forms of malignant disease, where higher Fgn levels occur in the plasma of patients bearing solid tumors, including carcinomas of the breast, compared to healthy controls.14 Fgn levels in the plasma may be as high as 10-12 µM in such individuals (compared to 4 µM in healthy individuals) and this may relate to tumor growth.15 However, levels may depend on the type of tumor present as van Wersch et al observed no significant difference between Fgn levels in patients with various gynecological tumors compared to healthy control groups.16

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Figure 1. The structure of fibrinogen and formation of fibrin. A) A schematic diagram of the tri-nodular structure of fibrinogen. The six polypeptide chains; 2 α- (black), 2 β- (dark gray) and 2 γ- (light gray) wrap around each other to form three globular domains, two D- and one E-. The fibrinopeptides (FpA and FpB) are found at the amino termini of the α- and β-chains within the E-domain. B) Thrombin cleaves the fibrinopeptides to release a fibrin monomer. These monomers join electrostatically before Factor XIIIa introduces cross-links between adjacent D-domains. C) Plasmin digestion of the fibrin polymer occurs in a stepwise manner and yields D-dimers, D-fragments from any non-covalently linked fibrin monomers, fibrin E-fragments and small peptides from the NH2-terminus of the β-chain and the COOH-terminus of the α-chains.

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Figure 2. In vitro effects of Fgn on: A) HDMEC migration across a collagen-coated filter in response to medium alone (control) or medium containing 10 ng/ml VEGF. B) HDMEC proliferation over a 48 hour period with VEGF, and C) Tubule formation by HDMEC on GFR- Matrigel in response to medium alone (control) or medium containing 10 ng/ml VEGF. A distribution curve is shown in D) indicating the distribution of individual tubule lengths in the absence (control) or presence of VEGF (10 ng/ml) or Fgn (1000 nM). Data shown in panels A-C are means + S.E.M. *P<0.005 compared to respective ‘no Fgn’ groups (Mann-Whitney U test).

Effects of Fibrinogen and Associated Peptide Fragments

Figure 3. In vitro effects of FnE on: A) HDMEC migration across a collagen-coated filter in response to medium alone (control) or medium containing 10 ng/ml VEGF. B) HDMEC proliferation over a 48 hour period with VEGF, and C) Tubule formation by HDMEC on GFR-Matrigel in response to medium alone (control) or medium containing 10 ng/ml VEGF. A distribution curve is shown in D) indicating the distribution of individual tubule lengths in the absence (control) or presence of VEGF (10 ng/ml) or FnE (100 nM). E) shows the increase in percentage cell death induced by a 6 hour exposure to FnE in medium alone or medium containing VEGF (10ng/ml). Data shown in panels A-C & E are means + S.E.M. *P<0.004 compared to respective ‘no FnE’ groups (Mann Whitney U test). 121

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Figure 4. Photomicrographs of tubules formed by HDMECs on GFR-Matrigel in vitro exposed to: I: Medium alone, II: 10 nM FnE, III: 100 nM FnE, IV: 1000 nM FnE for 6 h without VEGF. The lower doses of FnE stimulated tubule formation but 1000 nM FnE was seen to markedly inhibit this due to cytotoxicity.

Fgn has also been shown to extravasate from the leaky blood vessels in tumors, no doubt facilitated by VEGF-induced increases in vascular permeability, and then accumulate within the stroma of certain tumors, including invasive carcinomas of the breast. Studies have suggested that Fgn may increase the pro-angiogenic actions of VEGF by binding directly to this cytokine.17 As a number of receptors for Fgn exist on human endothelial cells, including the integrins αvβ3, α5β1,18-19 vascular endothelial cadherin (VE-Cadherin)20 and intercellular adhesion molecule-1 (ICAM-1),21 we investigated whether Fgn modified the responses of human endothelial cells to VEGF in vitro. Three important cellular events in angiogenesis are the migration, proliferation and differentiation of endothelial cells, all of which are stimulated by VEGF.22 Dejana et al showed that Fgn concentrations of up to 3 µM caused a marked increase in chemotaxis of endothelial cells in a dose-dependent manner.23 However this work was carried out in the absence of growth factors usually found in areas of angiogenesis, and used bovine pulmonary artery endothelial cells (BAEC). Further studies by Bull et al showed that increasing concentrations of Fgn (up to 250 nM in the medium) stimulated BAEC migration in a Boyden chamber assay.24 Fgn (250 nM) stimulated such a high level of cell migration that addition of the growth factors, epidermal growth factor (EGF) and platelet derived growth factor (PDGF), had no further effect.24 However, this work was limited as calf cells rather than human cells were used, and the effects of Fgn on VEGF-induced endothelial cell activity were not investigated. We have extended these reports by showing that Fgn stimulates migration of human dermal microvascular endothelial cells (HDMECs), the type of endothelial cell most closely related to tumor endothelial cells. Fgn (≥100 nM) stimulated migration through a collagen-coated filter in a Boyden chamber assay both in the absence and presence of VEGF (Fig. 2A). 25 However, it is not clear whether migration of the endothelial cells across the filter is dependent on a concentration gradient of Fgn (chemotactic activity) or whether it was simply due to an increase in cell motility (chemokinetic activity). Dejana et al using the same chamber model showed that when Fgn was placed in the bottom well the concentrations of Fgn in the wells above and below the filter reached equilibrium after 6 hours. 23 The assay used in the studies

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Figure 5. The production of Fibrinogen E-fragment. Plasmin cleavage of fibrinogen yields two D-fragments, one fibrinogen E-fragment and two small peptides from the amino-terminus of the β-chains, β1-42.

described in this chapter assessed migration after 4.5 hours with Fgn in the bottom wells, and therefore it is possible that a concentration gradient of Fgn across the membrane remained at the end of the assay. The work presented by Dejana et al using BAEC, showed a maximal migratory response in the presence of a positive Fgn gradient, but also with Fgn on both sides of the membrane, suggesting that Fgn has both chemotactic (movement along a concentration gradient) and chemokinetic (increase in cell motility) activity. 23 Together, these findings suggest that where Fgn levels are high, such as in and around activated endothelium, it is likely to promote endothelial cell migration. As directed migration is an important part of the angiogenic process, this data indicates that Fgn has the potential to be an angiogenic protein and to potentiate the endothelial activation induced by VEGF. We have also shown that Fgn (1000 nM) increases proliferation of HDMEC, using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay in both the absence and presence of VEGF (10 ng/ml) (Figure 2B is with VEGF). However, these results contrast with those reported by Sahni et al who demonstrated that Fgn did not increase the proliferation of endothelial cells in response to VEGF.17 This could be due to differences in the type of endothelial cells (HDMEC vs human umbilical vein endothelial cells, HUVEC) and the concentrations of growth factors (10 ng/ml vs 20 ng/ml) and Fgn (1000 nM vs 30.3 nM) used in these two studies. Plating cells on growth factor reduced (GFR-) Matrigel mimics the early events in the differentiation process as the endothelial cells begin to migrate, elongate and round up to form a lumen.26 Our studies demonstrated that Fgn significantly increased tubule formation at ≥100 nM, with elevated tubule number and area, both in the absence and presence of VEGF (Fig. 2C). In addition VEGF increased the length of individual tubules causing an increase in total tubule length per field of view (Fig. 2D). Although Fgn increased the number of tubules and total area, it did not increase the length of individual tubules, indicating that Fgn stimulates this process by different mechanisms when compared to VEGF. Addition of both Fgn and VEGF to the cells appeared to cause an additive effect on tubule formation suggesting that the combined presence of these factors in a tumor may cause greater stimulation of angiogenesis than either alone.

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Figure 6. The structure of A: FgnE and B: FnE. These two fragments differ in only in the presence or absence of the amino-terminus of the alpha chains of FgnE (as indicated by the circle).

Fgn has recently been shown to bind to VEGF in vitro in a manner that does not interfere with the growth factor activity.17 Binding to Fgn may help to present the growth factor to endothelial cells to allow maximum activation of the endothelial cells.17 This may be a possible explanation for the additive effect observed, when endothelial cells are treated with VEGF and Fgn combined in the migration, proliferation and tubule formation assays compared to VEGF or Fgn alone (Fig. 2). In pathological tissues, such as malignant tumors, the stimulation of endothelial cells is likely to be mediated by VEGF, amongst other growth factors. VEGF also stimulates the permeability of blood vessels to plasma proteins including Fgn, thereby enhancing the accumulation of this protein within areas of angiogenesis. It is clear from the data presented that Fgn may stimulate angiogenesis and enhance the effects of VEGF. Therefore, in areas of Fgn deposition within tumors, Fgn is likely to provide an additional stimulus for the promotion of angiogenesis.

Fibrin, VEGF and Angiogenic Mechanisms Fibrin has been shown to accumulate in the majority of angiogenesis-driven conditions. Fibrin deposition has long been associated histologically with endothelial cell injury in vivo and is a characteristic feature of both solid tumor growth and wound healing. This suggests that fibrin-endothelial cell interactions not only occur in vivo but may also be extremely important in regulating vascular function as well as providing the initial matrix required for the formation of granulation tissue in wounds. Fibrin deposition is also found in other disease states where angiogenesis is important, such as within atherosclerotic plaques.27 The formation and deposition of fibrin within both human and experimental tumors is thought to be of importance in the local host defence response.28 It is more likely, however, that the fibrin gel serves as a provisional matrix to facilitate the entry of fibroblasts and macrophages (and thus the deposition and re-modelling of the stromal compartment) as well as the ingrowth of new blood vessels.29-31 Endothelial cells have been shown to line up along the planar surface of fibrin, and form tubules, which infiltrate the fibrin structures, both in vitro and in vivo.32-33 This suggests that fibrin itself forms a structure that allows the formation of new vessels both by providing a scaffold for the formation of new vessels and by the degradation of the fibrin polymer, which releases growth factors, including VEGF, that have become sequestered into the matrix. These growth factors then stimulate endothelial cell migration and differentiation, resulting in the formation of new vessels. Fibrin gels implanted subcutaneously onto the guinea pig dorsum become vascularized with new vessels that are fully functional. Addition of growth factors to the gels, including

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Figure 7. In vitro effects of FgnE on: A) HDMEC migration across a collagen-coated filter in response to medium alone (control) or medium containing 10 ng/ml VEGF. B) HDMEC proliferation over a 48 hour period with VEGF, and C) Tubule formation by HDMEC on GFR-Matrigel in response to medium alone (control) or medium containing 10 ng/ml VEGF. D) Shows the increase in percentage cell death induced by a 6 hour exposure to FnE in medium alone or medium containing VEGF (10 ng/ ml). All data shown are means + S.E.M. *P<0.002 compared to respective ‘no FgnE’ groups (Mann Whitney U test). Panel A reproduced with permission from Bootle-Wilbraham CA et al., Cancer Research 2000; 60: 4719-4724. 2000 AACR.

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Figure 8. Photomicrographs of tubules formed by HDMECs grown on GFR-Matrigel in vitro, with or without 10 ng/ml VEGF and in the absence (‘no protein’) or presence of different concentrations of human FgnE for 6h.

VEGF and other mitogens, stimulate an increased level of angiogenesis suggesting that although fibrin alone stimulates angiogenesis, the presence of other factors in wounds further stimulate angiogenesis. 33 VEGF has also been shown to bind to fibrin, which may, in a similar manner to Fgn, protect the growth factor from proteolytic degradation and present the VEGF to the endothelial cells in a way which causes maximum activation of the endothelial cells.17 Together, these findings suggest that the formation of fibrin within tumors may enhance the activation of endothelial cells in response to VEGF.

Stimulation of VEGF Induced Angiogenesis by Fibrin E-Fragment Patients with tumors show unusually high levels of plasma fibrin production in addition to high levels of fibrinolysis, due to an increased production of plasmin.34 The final products of fibrinolysis are the Fibrin E-fragment (FnE) and associated D-dimers (Fig. 1C). D-dimer levels

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Figure 9. Effect of pre-exposure of HDMEC to FgnE for 1 hour on: A) Migration by HDMECs in vitro. Mean ± SEM number of cells migrating across a collagen-coated filter in response to control medium or medium containing VEGF (10 ng/ml). The cells were either exposed to varying concentrations of FgnE for an hour prior to the assay and then washed and placed in the assay in the absence of the protein (‘pre-exposed’), or the same concentrations of FgnE were placed in the lower chamber for the duration of the assay alone. *P<0.01 compared to respective control (no FgnE) group. ^P<0.04 compared to respective non pre-exposed group (Mann Whitney U test). B) Tubule formation of HDMEC on GFR-Matrigel in the absence or presence of VEGF (10 ng/ml). Mean ± S.E.M. area of tubule formation per field of view. Again, cells were either exposed to varying concentrations of FgnE for an hour prior to the assay or during the assay. *P<0.02 compared to respective control (no FgnE group). ^P<0.001 compared to respective non pre-exposed group (Mann Whitney U test).

are also elevated in cancer patients, indicating a fast turnover of fibrin.35 Angiogenesis is known to require the degradation of the extracellular matrix (ECM) including fibrin. This suggests that the breakdown of this protein is involved in angiogenesis, and that some fibrin degradation

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products may be potential pro-angiogenic candidates due to their production in areas of high angiogenic activity, such as in wound healing, atherosclerotic plaques and at the invasive edge of such carcinomas such as breast, prostate and pancreas.36-39 Thompson and co-workers showed that fibrin degradation products (FDP) stimulated cell proliferation including angiogenesis in the chick chorioallantoic membrane (CAM) assay.36 The results published in a subsequent paper showed that FDP at a concentration of 245 µg/ml stimulated DNA synthesis and produced a 44% increase in mesenchymal layer blood vessels in the CAM assay.40 When FDP preparations were run down anti-E-fragment antibody columns, there was a reduction in angiogenesis stimulated by FDP,41 suggesting that the FDP activity resided in the E-fragment. To test this hypothesis, FnE was extracted from plasmin digests of fibrin and was subsequently shown to stimulate angiogenesis in the CAM assay.40 Commercial fibrinogen E-fragment (FgnE) was inactive in their assays, although treatment with thrombin to remove the fibrinopeptides, and produce FnE, resulted in a molecule capable of stimulating angiogenesis.40 This suggested that the generation of angiogenic activity within fragment E is thrombin dependent, requiring the removal of fibrinopeptides. This either exposes the active site at the amino termini of the α-chains of FnE and/or changes the conformation of this protein to reveal the active site. Furthermore, recent data from the same authors have demonstrated that FDP also enhances the outgrowth of smooth muscle cells (SMC) from rabbit aortic explants in culture,42 which is inhibited in the presence of anti-FnE antibodies indicating that FnE is mitogenic for SMC.42 This data suggests that FnE may play a role in the maturation of vessels in addition to stimulating angiogenesis. However, Thompson’s work was limited in that it investigated the effects of human FnE on non-human rather than human endothelial cells.36,40-42 We therefore extended these studies, and showed that FnE (≥ 10 nM) stimulated the migration of HDMEC in vitro in a dose dependent manner, both in the presence or absence of VEGF (Fig. 3A).43 However, although the highest dose of FnE tested (1 µM) appeared to inhibit both the migration (Fig. 3A) and proliferation of HDMEC (Fig. 3B in the presence of VEGF ) this concentration of the fragment was cytotoxic to endothelial cells, (Fig. 3E). This suggests that the higher the local concentration of this FDP in a given tissue (within the limits found naturally in the body i.e. up to 330 nM) the greater the migration of endothelial cells with a potential increase in the rate of new blood vessel formation up to the limits demonstrated to be cytotoxic to endothelial cells. At non-cytotoxic levels (<1000 nM FnE) an additive effect is seen in all the assays performed when endothelial cells are treated with VEGF and FnE in combination compared to either alone. FnE (10 and 100nM) markedly enhanced tubule formation on GFR-Matrigel in vitro, both in the absence and presence of VEGF, as evidenced by an increase in both tubule area and number (Figs. 3C and 4 (minus VEGF), respectively).43 VEGF increased tubule length, a process mimicked by FnE (Fig. 3D). This suggests that by binding to its receptor, FnE may trigger similar cellular responses to those involved in VEGF-induced differentiation. However, the effect of FnE alone on the proliferation, migration and differentiation of HDMECs is less marked that that seen with equimolar concentrations of VEGF (10 ng/ml VEGF = 0.6 nM), suggesting that this protein may not activate HDMECs via the same signaling pathway as VEGF, or at least not with the same efficacy. When in vivo conditions of wound healing are considered, however, FnE is likely to be more abundant than VEGF at a site of continuous fibrin degradation. VEGF levels in normal human plasma are 0.9 pM and are elevated in cancer patients up to a maximum level of 12 pM.44 Endogenous tissue levels of FDP are likely to be increased (usually in the range of 200-300 nM) at disease sites in vivo.42 When compared to VEGF this may indicate that, as FnE is less potent at promoting angiogenesis, where FnE is naturally found it may enhance VEGF stimulated angiogenesis, rather than promoting angiogenesis alone. Currently the receptor for FnE on endothelial cells is unknown. FnE does not contain the sequences involved in the receptor binding of fibrin or Fgn. Fgn contains arginine- glycine-

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aspartic acid (RGD) sequences at the carboxy terminus of the α-chain, which bind to the integrins αvβ3 and α5β1 present on endothelial cells.18-19 Neither this sequence nor the sequences for binding to the other endothelial cell receptors for Fgn, namely the γ-chain sequence for binding ICAM-1 (γ117-133)21 and the amino terminus of the β-chain, β15-42 (for binding to VE-Cadherin)20 are found in FnE, as these are cleaved by plasmin in the production of this fragment. This suggests that FnE may stimulate endothelial cell activity via novel, as yet uncharacterized receptor(s). We conclude that the enzymatic degradation of fibrin (e.g., during the resolution of blood clots) to form its various breakdown products is likely to promote angiogenesis within the surrounding tissues due to the formation of the fragment, FnE.

Fibrinogen E-Fragment Inhibits VEGF Activation of Endothelial Cells Fibrinogen E-fragment (FgnE) is produced when plasmin cleaves Fgn (Fig.5) and differs from the FnE fragment discussed in the previous section in the structure of the α-chains as shown in Figure 6. FgnE, contains intact α-chains and amino termini whereas the production of FnE leads to the cleavage of the amino termini by the action of thrombin. The only previous suggestion of a role for FgnE in the regulation of angiogenesis came from Dejana et al23 This group established that FgnE could inhibit Fgn-stimulated chemotaxis of bovine endothelial cells (BAEC) in vitro and as chemotaxis is an important component of the angiogenesis pathway, it is possible that FgnE induces opposing effects to FnE in vitro. However, this work was carried out in the absence of growth factors, usually found in areas of angiogenesis, especially within the tumor microenvironment (including VEGF) and could be interpreted as indicating that FgnE inhibits Fgn-stimulated chemotaxis due to the 2 molecules competing for the same binding site on endothelial cells. We extended this report by showing that FgnE (≥ 10 nM) inhibits the migration and tubule formation by HDMEC stimulated by VEGF in a dose-dependent manner (Fig.s 7A and C, respectively and 8).25 It should be noted that HDMEC migration is only reduced by FgnE in the presence of VEGF, whereas both ‘basal’ and VEGF-stimulated tubule formation are inhibited by this FgnE. The degree of inhibition, however, is greater in the presence of VEGF, and within the latter assay cells are not completely quiescent in the absence of VEGF as they are stimulated by the residual levels of growth factors remaining in the GFR-Matrigel. Together, our FgnE data suggest that, where endothelial cells are activated by VEGF in areas of angiogenesis, the presence of FgnE may inhibit this process. We also found that FgnE inhibits similar bFGF-activated endothelial cells in vitro (data not shown),25 suggesting that FgnE may potentially be of use as a broad-acting, anti-angiogenic agent. Anti-angiogenic agents often have a significant effect on endothelial proliferation, such as reducing the rate or inhibiting the process completely.45 However, FgnE failed to inhibit HDMEC proliferation at the lower two doses tested (10 and 100 nM) in either the presence or absence of VEGF (Fig. 7B in the presence of VEGF). At the highest dose tested, (1000 nM - as with FnE) FgnE consistently showed inhibition of HDMEC proliferation. This was due, at least in part, to the cytotoxic effect of FgnE at this dose (Fig. 7D). The presence of VEGF, however, does not prevent the cytotoxic effect of 1000 nM FgnE and the same levels of cytotoxicity are seen in the presence and absence of the growth factor. As VEGF has been shown to bind to both Fgn and fibrin17 and the binding site for this interaction on Fgn is not yet known, it is possible that FgnE may mediate its effects by binding to VEGF in these assay systems and preventing VEGF from binding to its receptors on the cells. However, experiments carried out by Sahni et al have shown that binding of VEGF to Fgn does not prevent VEGF from activating endothelial cells, suggesting that it is unlikely that binding to FgnE would cause inhibition.17 Moreover, we have shown that exposure of HDMEC to FgnE (≥ 10 nM) for one hour prior to addition of the cells to the migration or GFR-Matrigel assays in vitro (which were then run in the absence of FgnE), was sufficient to significantly inhibit migration and tubule formation in response to VEGF (Fig. 9).

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The Effects of Other Fibrinogen/Fibrin Related Fragment on VEGF-Activation of Endothelial Cells The effects of the remaining fragments cleaved from Fgn in the production of fibrin, namely fibrinopeptide A and B, and the fragments generated when Fgn or fibrin are degraded by plasmin, namely α-chain C-terminus, β1-42, β15-42, fibrinogen D-fragment and D-dimer, on angiogenic processes are not known at present. The fibrinopeptides (FpA and FpB) have been shown to influence neutrophil migration and de-granulation,46-47 but do not have any reported effects on angiogenesis. Indeed our studies have shown that neither fibrinopeptides A or B effect migration, proliferation or differentiation in vitro, in either the absence or presence of VEGF (Staton CA and Lewis CE, unpublished data). 43 The cleavage of fibrinopeptide B from Fgn by the action of thrombin exposes β15-42, still attached to the fibrin monomer, which has been shown to be the portion of fibrin responsible for promoting endothelial cell spreading, and proliferation.20, 48-49 However, the effects of the two peptides released when Fgn or fibrin are cleaved by plasmin, namely β1-42 and β15-42 have not been investigated fully, although it is known that they bind to VE-cadherin, the receptor for the β15-42 sequence of fibrin that is involved in fibrin-mediated angiogenesis. We have also shown that fibrinogen D-fragment (FgnD) has no effect on the migration, proliferation and differentiation of endothelial cells in vitro (Staton CA & Lewis CE, unpublished data). In contrast, it has been reported that FgnD can disorganize endothelial cell monolayers, causing the cells to retract from one another and to become detached from the substratum.50 However, a more recent study has shown that FgnD causes an increase in cell permeability without cell detachment or lysis.51 Although the effects of FgnD on the permeability of endothelial cells in the presence of VEGF has not been investigated it is possible that FgnD may serve to enhance VEGF-induced permeability by stimulating either the same or an alternative pathway. In contrast there are no reports of D-dimer or the α-chain C-terminal region having any effect on endothelial cells or on the activity of VEGF.

Conclusion Within the microenvironment of a solid tumor it can be seen that there is considerable potential for Fgn and its breakdown products to modulate the pro-angiogenic functions of VEGF. In vivo VEGF stimulates endothelial cells rendering them permeable, allowing the plasma Fgn to accumulate in the tumor stroma. This is likely to promote an angiogenic phenotype by co-stimulating endothelial cells and enhancing VEGF activity by binding VEGF and/or by acting directly on the cells. In many tumors the accumulated Fgn is cleaved by thrombin to form fibrin, which also acts in conjunction with VEGF to stimulate endothelial cell migration and tubule formation. The fibrin also provides a temporary matrix, which aids the formation of the tumor stroma and provides a scaffold for blood vessel formation. Degradation of fibrin by plasmin causes release of fibrin E fragment which also stimulates angiogenesis. In contrast plasmin degradation of Fgn (which is rare in the body) generates the fragment, FgnE, which inhibits endothelial cell activation both in the presence and absence of VEGF. As the production of plasmin is tightly regulated, occurring only in areas of fibrin deposition, and fibrin itself acts as a co-factor which increases the catalytic efficiency of plasmin production from plasminogen, the production of FgnE is rare in vivo.52 Thus it is most likely that Fgn, fibrin and FnE will be found in areas of angiogenesis in vivo where they serve to enhance the activity of VEGF.

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References 1. Browder T, Folkman J, Pirie-Shepherd S. The hemostatic system as a regulator of angiogenesis. J Biol Chem 2000; 275:1521-24. 2. Francis JL. Fibrinogen, fibrin stabilization and fibrinolysis; clinical, biochemical and laboratory aspects. Ellis Horwood Series in Biomedecine Chichester, 1988. 3. Brown JH, Volkmann N, Jun G et al. The crystal structure of modified bovine fibrinogen. Proc Natl Acad Sci USA 2000; 97:85-90. 4. Yang Z, Mochalkin I, Veerapandian L et al. Crystal structure of native chicken fibrinogen at 5.5-A resolution. Proc Natl Acad Sci USA 2000; 97:3907-3912. 5. Hermans J, McDonagh J. Fibrin: Structure and interactions. Semin Thromb Hemost 1982; 8:11-24. 6. Blomback B, Blomback M. The molecular structure of fibrinogen. Ann NY Acad Sci 1972; 202:77-97. 7. Ferry JD. The mechanisms of polymerization of fibrin. Proc Natl Acad Sci USA 1952; 38:566-569. 8. Fowler WE, Erickson HP, Hantgan RR et al. Cross-linked fibrinogen dimers demonstrate a feature of the molecular packing in fibrin fibers. Science 1981; 211:287-289. 9. Marder VJ, Shulman NR, Carroll WR. High molecular weight derivavtives of human fibrinogen produced by plasmin. I. Physicochemical and immunological characterization. J Biol Chem 1969; 244:2111-2119. 10. Lucas MA, Straight DL, Fretto LJ et al. The effects of fibrinogen and its cleavage products on the kinetics of plasminogen activation by urokinase and subsequent plasmin activity. J Biol Chem 1983; 258:12171-12117. 11. Francis CW, Marder VJ. Heterogeneity of normal human fibrinogen due to two high molecular weight variant gamma chains. Ann NY Acad Sci 1983; 408:118-120. 12. Francis CW, Marder VJ, Barlow GH. Plasmic degradation of crosslinked fibrin. Characterization of new macromolecular soluble complexes and a model of their structure. J Clin Invest 1980; 66:1033-1043. 13. Gaffney PJ, Joe F. The lysis of crosslinked fibrin by plasmin yields initially a single molecular complex, D dimer-E. Thromb Res 1979; 15:673-687. 14. Nagy I, Losonczy, H. Hemostatic alterations in lymphomas and tumors. Acta Med Hung 1987:71-82. 15. Lu DY, Chen XL, Cao JY et al. Effects of cancer chemotherapy on the blood fibrinogen concentrations of cancer patients. J Int Med Res 2000; 28:313-7. 16. van Wersch JW, Peters C, Ubachs JM. Coagulation factor XIII in plasma of patients with benign and malignant gynaecological tumors. Eur J Clin Chem Clin Biochem 1994; 32:681-684. 17. Sahni A, Francis CW. Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation. Blood 2000; 96:3772-3778. 18. Suehiro K, Gailit J, Plow EF. Fibrinogen is a ligand for integrin α5β1 on endothelial cells. J Biol Chem 1997; 272:5360-5366. 19. Yokoyama K, Zhang X, Medved L et al. Specific binding of integrin αvβ3 to the fibrinogen γ and αE chain C-terminal domains. Biochem 1999; 38:5872-5877. 20. Francis CW, Bunce LA, Sporn LA. Endothelial cell responses to fibrin mediated by FPB cleavage and the amino terminus of the beta chain. Blood Cells 1993; 19:291-306. 21. Pluskota E, D’Souza SE. Fibrinogen interactions with ICAM-1 (CD54) regulate endothelial cell survival. Eur J Biochem 2000; 267:4693-4704. 22. Ortega N, L’Faqihi FE, Plouet J. Control of vascular endothelial growth factor angiogenic activity by the extracellular matrix. Biol Cell 1998; 90:381-390. 23. Dejana E, Languino LR, Polentarutti N et al. Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding. J Clin Invest 1985; 75:11-18. 24. Bull DA, Seftor EA, Hendrix MJC et al. Putative vascular endothelial cell chemotactic factors: Comparison in a standardized migration assay. J Surg Res 1993; 55:473-479. 25. Bootle-Wilbraham CA, Tazzyman S, Marshall JM et al. Fibrinogen E-fragment inhibits the migration and tubule formation of human dermal microvascular endothelial cells in vitro. Cancer Res 2000; 60:4719-4724. 26. Shen J, Ham RG, Karmiol S. Expression of adhesion molecules in cultured human pulmonary microvascular endothelial cells. Microvasc Res 1995; 50:360-372. 27. Smith EB, Thompson WD. Fibrin as a factor in atherogenesis. Thromb Res 1994; 73:1-19. 28. Colucci M, Giavazzi R, Allessandri G et al. Procoagulant activity of sarcoma sublines with different metastatic potential. Blood 1981; 57:733-735. 29. Dvorak HF, Senger DR, Dvorak AM. Fibrin as a component of the tumor stroma: origins and biological significance. Cancer Metastasis Rev 1983; 2:41-73.

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30. Dvorak HF, Nagy JA, Berse B et al. Vascular permeability factor, fibrin, and the pathogenesis of tumor stroma formation. Ann N Y Acad Sci 1992; 667:101-111. 31. Nagy JA, Meinwald YC, Scheraga HA. Immunochemical determination of conformational equilibria for fragments of the Aα chain of fibrinogen. Biochem 1982; 21:1794-1806. 32. Nehls V, Schuchardt E, Drenckhahn D. The effects of fibroblasts, vascular smooth muscle cells, and pericytes on sprout formation of endothelial cells in a fibrin gel angiogenesis system. Microvasc Res 1994; 48:349-363. 33. Dvorak HF, Harvey VS, Estrella P et al. Fibrin containing gels induce angiogenesis. Lab Invest 1987; 57:673-686. 34. Wojtukiewicz MZ, Sierko E, Klement P et al. The hemostatic system and angiogenesis in malignancy. Neoplasia 2001; 3:371-384. 35. Sagripanti A, Carpi A, Baicchi U et al. Plasmatic parameters of fibrin formation and degradation in cancer patients: correlation between fibrinopeptide A and D-dimer. Biomed & Pharmacother 1993; 47:235-239. 36. Thompson WD, Smith EB, Stirk CM et al. Factors relevant to stimulatory activity of fibrin degradation products in vivo. Blood Coagul Fibrinolysis 1990; 1:517-520. 37. Costantini V, Zacharski LR. Fibrin and cancer. Thromb and Hemost 1993; 69:406-414. 38. Braun JS, Novak R, Torzewski M et al. Intracerebral hemorrhages, fibrinolysis and prostate carcinoma. J Neurol 2002; 249:478-479. 39. Wojtukiewicz MZ, Rucinska M, Zacharski LR et al. Localisation of blood coagulation factors in situ in pancreatic carcinoma. Thromb Hemost 2001; 86:1416-1420. 40. Thompson WD, Smith EB, Stirk CM et al. Angiogenic activity of fibrin degradation products is located in fibrin fragment E. J Pathol 1992; 168:47-53. 41. Thompson WD, Smith EB, Stirk CM et al. Atherosclerotic plaque growth: presence of stimulatory fibrin degradation products. Blood Coagul Fibrinol 1990; 1:489-493. 42. Naito M, Stirk CM, Smith EB et al. Smooth muscle cell outgrowth stimulated by fibrin degradation products: the potential role of fibrin fragment E in restenosis and atherogenesis. Thromb Res 2000; 98:749-757. 43. Bootle-Wilbraham CA, Tazzyman S, Thompson WD et al. Fibrin E fragment stimulates the proliferation, migration and differentiation of human microvascular endothelial cells in vitro. Angiogenesis 2001; 4:269-275. 44. Dirix LY, Vermeulen PB, Pawinski et al. Elevated levels of the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in the sera of cancer patients. Br J Cancer 1997; 76:238-243. 45. Auerbach W, Auerbach R. Angiogenesis inhibition: a review. Pharmac Ther 1994; 63:265-311. 46. Senoir RM, Skogen WF, Griffin GL et al. Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B. J Clin Invest 1986; 77:1014-1019 47. Wojtecka-Lukasik E, Maslinski S. Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation. J Physiol Pharmacol 1992; 43:173-181. 48. Sporn LA, Bunce LA, Francis CW. Cell proliferation on fibrin: Modulation by fibrinopeptide cleavage. Blood 1995; 86:1802-1810. 49. Bunce LA, Sporn LA, Francis CW. Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain. J Clin Invest 1992; 89:842-850. 50. Dang CV, Bell WR, Kaiser D et al. Disorganisation of cultured vascular endothelial cell monolayers by fibrinogen fragment D. Science 1985; 227:1487-1490. 51. Ge M, Ryan TJ, Lum H et al. Fibrinogen degradation product fragment D increases endothelial monolayer permeability. Am J Physiol 1991; 261:L283-9. 52. Camiolo SM, Thorsen S, Astrup T. Fibrinogenolysis and fibrinolysis with tissue plasminogen activator, urokinase, streptokinase activated human globulin, and plasmin. Proc Soc Exp Biol Med 1971; 138:277-280.

CHAPTER 13

Vascular Endothelial Growth Factor (VEGF) and Its Role in Non-Endothelial Cells: Autocrine Signalling by VEGF Angela M. Duffy, David J. Bouchier-Hayes and Judith H. Harmey

Abstract

V

ascular endothelial growth factor (VEGF) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. VEGF is up-regulated in many tumors and its contribution to tumor angiogenesis is well defined. In addition to endothelial cells, VEGF and VEGF receptors are expressed on numerous non-endothelial cells including tumor cells. This review examines the relevance of VEGF signalling in non-endothelial cells and explores the probable mechanisms involved. The existence of autocrine VEGF signalling pathways in tumor cells is discussed in relation to anti-VEGF anti-tumor strategies now being developed.

Introduction Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), was originally described as an endothelial cell-specific mitogen.1 VEGF is produced by many cell types including tumor cells,2,3 macrophages,4 platelets,5 keratinocytes,6 and renal mesangial cells.7 The activities of VEGF are not limited to the vascular system; VEGF plays a role in normal physiological functions such as bone formation,8 hematopoiesis,9 wound healing,10 and development.11 Anti-VEGF strategies to treat cancers were designed to target the pro-angiogenic function of VEGF and thereby inhibit neovascularization. However, anti-VEGF therapies may have a dual effect since evidence is accumulating to support the existence of both paracrine and autocrine VEGF loops within tumors. It has been suggested that direct stimulation of tumor cells by VEGF may protect the cells from apoptosis and increase their resistance to conventional chemotherapy and radiotherapy.12 Chemotherapy and radiotherapy have been shown to increase VEGF within tumors,13 and this increased VEGF may in fact protect tumor cells from these interventions. Anti-VEGF therapies are therefore likely to target both the pro-angiogenic activity of VEGF and the anti-apoptotic/pro-survival functions of VEGF. Combination therapies using anti-VEGF therapies with chemotherapy and/or radiotherapy are effective against many types of tumor, possibly because in addition to angiogenesis inhibition, VEGF blockade renders tumor cells more susceptible to conventional treatment. This chapter reviews the evidence for VEGF autocrine signalling in non-endothelial cells, including tumor cells.

VEGF in the Cardiovascular System VEGF plays an important role within the cardiovascular system. Recently VEGF expression has been demonstrated in cardiac myofibroblasts, non-endothelial cells with the VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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morphological features of fibroblasts. Myofibroblasts play a major role in the growth, development and repair of normal tissue and are found at the site of infarction. RT-PCR, Northern blot and Western blot analysis confirmed the presence of mRNA and protein for VEGF, VEGFR-1 (VEGF receptor 1 also known as Flt-1, fms-like tyrosine kinase) and VEGFR-2 (KDR/Flk-1, Fetal liver kinase 1 is the murine homologue of human Kinase insert Domain-containing Receptor) in rat myofibroblasts isolated from heart infarcts.10 Coexpression of VEGF and its receptors on the myofibroblasts suggests that VEGF contributes to tissue remodelling at the site of infarction in an autocrine manner. VEGF may also play a role in atherosclerosis.14 Uptake of oxidised low-density lipoprotein (ox-LDL) by macrophages initiates foam cell formation in atherosclerotic lesions.15 Oxidised LDL increased VEGF production by the U937 monocyte cell line14 and by macrophages.16 As VEGF also increases vascular permeability, VEGF production by foam cells and macrophages may aggravate atherosclerosis by enhancing vessel permeability to LDL.

VEGF and the Central Nervous System (CNS) In the central nervous system (CNS) both positive (pro-migratory) and negative (anti-migratory) regulatory factors are essential for axonal guidance.17 Following prolonged exposure, Sema3A, a member of the semaphorin family, acts as an inhibitor of neuronal migration and induces neuronal cell death18 through the neuropilin-1 receptor (NP-1).19 However, in addition to Sema3A binding, NP-1 also acts as an additional receptor for VEGF165 isoform.20 The relationship between Sema3A and VEGF was explored in Dev cells,21 undifferentiated cells derived from a cerebellar medullablastoma that behave as pluripotential neural progenitor cells.22 NP-1 mRNA expression was detected in Dev cells by RT-PCR and in situ hybridization. Western blotting and immunohistochemical analysis confirmed that NP-1 was expressed on the cell surface. VEGF165 or anti-NP-1 antibody blocked the effect of Sema3A on these cells, suggesting that VEGF165 binds competitively to NP-1 to block Sema3A signalling. VEGF165 stimulated migration and promoted proliferation of the Dev cells whereas Sema3A binding inhibited cell migration and induced cell death. A recent paper has shown that the Sema3A/VEGF balance in breast carcinoma cells determines the chemotactic rate of the tumor cells towards conditioned NIH3T3 medium.23 Breast carcinoma cell lines with the lowest chemotatic rate have the highest ratio of Sema3A to VEGF. Overexpression of VEGF by tumor cells may enhance tumor cell migration via inhibition of the Sema3A/ NP-1 pathway. Dev cells also expressed VEGFR-1 and blockade of VEGFR-1 reduced the inhibition of neuronal cell migration by Sema3A.21 It appears that both NP-1 and VEGFR-1 are required for Sema3A activity in these neuronal cells. NP-1 binds with high affinity to VEGFR-1.24 NP-1 has a very short intracellular domain and appears to require a coreceptor to transduce a signal20 thus, VEGFR-1 may serve as a coreceptor for NP-1 in the modulation of Sema3A signalling. Both VEGF121 and VEGF165 inhibited Sema3A-induced apoptosis, and at higher concentrations reduced apoptosis below basal levels indicating an additional neuroprotective effect. VEGF is induced in many CNS pathologies where it may have a neuroprotective role. VEGF has a neurotrophic effect and enhances survival of Schwann cells,25 and protects hippocampal neurons from ischemic injury.26 Impaired VEGF induction in the spinal cord results in motor neuron degeneration.27 In addition, when cerebellar granule neurons (CGNs) were exposed to 5% hypoxia for 9 hours VEGF, VEGFR-1 and VEGFR-2 expression increased, and a neutralizing antibody to VEGF, DC 101, inhibited hypoxic preconditioning.28 Thus, VEGF autocrine or paracrine mechanisms appear to play a role in CGN cell survival following hypoxic preconditioning. In CGNs Akt (also known as Protein Kinase B/ PKB) was phosphorylated in response to VEGF and other studies have shown that VEGF stimulation in neurons is linked to PI3-K (Phosphatidylinositol 3'-kinase) and Akt activation and neuronal protection.29

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VEGF and Its Role in Bone Although cartilage is essentially an avascular tissue, neovascularization does occur in the growth plate of developing bone.30 VEGF is produced by hypertrophic chondrocytes in the growth plate where it co-ordinates extracellular matrix (ECM) remodelling, angiogenesis, and bone formation.8 VEGF is expressed in the synovial fluid of patients with rheumatoid arthritis.31 and the cartilage of patients with osteoarthritis (OA).32 VEGF is found in normal cartilage but only osteoarthritic cartilage expresses the VEGF receptors, VEGFR-1, VEGFR-2, and NP-1. The level of VEGF in the culture media from OA chondrocytes was 3.3-fold greater than in media from normal chondrocytes. These results suggest that autocrine and/or paracrine signalling by VEGF may play a role in the pathology of osteoarthritis.

VEGF in Hematopoietic Cells and Hematological Malignancies

VEGF plays a central role in hematopoiesis.9 VEGF is expressed in the bone marrow and cytokine stimulation of hematopoietic stem cells (HSCs) greatly increases VEGF levels within these cells.33 Both VEGFR-1 and VEGFR-2 are expressed on HSCs, 34,35 and VEGFR-2 has been identified as a positive functional marker for pluripotent HSCs.36 Elevated levels of VEGF and VEGFR-2 in young mice resulted in mobilization and recruitment of HSCs to the spleen during vascular remodelling.37 VEGF and VEGFR-2 gene knockout resulted in embryonic lethality due to impaired hematopoiesis and angiogenesis.38 VEGF inhibited maturation of antigen-presenting cells, dendritic cells and many other hematopoietic cells in vivo.39 HSC survival is controlled by an internal autocrine VEGF loop.40 VEGF is coexpressed with its receptors on hematopoietic cells, suggesting that autocrine mechanisms are involved in the regulation of hematopoiesis. The ability of VEGF-deficient HSCs to form colonies in vitro was dramatically reduced, with most cells developing characteristics associated with apoptosis.40 The survival of leukemic cells in serum-free conditions is also dependent on an autocrine VEGF/VEGFR-2 loop.41 In vivo studies using mice inoculated with human HL-60 leukemic cells revealed that both paracrine and autocrine VEGF signalling pathways were necessary for tumor cell survival.42 Neutralizing antibodies to murine VEGFR-2, which targeted only the paracrine VEGF signalling in the host endothelial cells, prolonged mouse survival but did not eradicate the disease. However, coadministration of the murine antibodies with antibodies that neutralized human VEGFR-2 and inhibited autocrine VEGF signalling in the human leukemic cells of the tumor had a synergistic effect on the survival of the inoculated mice. Blocking both the paracrine and autocrine VEGF loops within the tumor decreased leukemia invasiveness and resulted in prolonged remission in 40% of the animals. Neutralizing antibodies to human and murine VEGFR-1 had no notable effect in this model, demonstrating that autocrine and paracrine VEGF/VEGFR-2 signalling pathways play an important role in leukemia proliferation and engraftment in vivo. VEGF and its receptors, VEGFR-1 and VEGFR-2, are overexpressed in many human hematopoietic tumor cell lines,43 and in bone marrow failure states such as chronic myleomonocytic leukemia and acute myelogenous leukemia.44,45 In a study of patients with multiple myeloma, VEGF, VEGFR-1 and VEGFR-2 were detected in 78% of the bone marrow samples examined.46 VEGF production has been correlated with disease progression in patients with a variety of hematological malignancies.47 In addition to expressing VEGF receptors on the cell surface, some leukemic cells also produce a VEGF antagonist. Soluble Flt-1/VEGFR-1 (sFlt-1) has been detected in NALM-16 and P30/OHKUBO hematopoietic cell lines,48 derived from patients with ALL.49,50 RT-PCR analysis identified sFlt-1 mRNA expression in an additional fifteen hematopoietic cell lines. sFlt-1 purified from NALM-16 cells bound to VEGF with high affinity, and cell supernatants from NALM-16 and P30/OHKUBO cell lines reduced VEGF production in KATO-III cell cultures and inhibited VEGF-dependent growth of human umbilical vein endothelial cells (HUVECs).48

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VEGF Signalling in Hematopoietic Cells VEGF has been shown to inhibit nuclear factor kappa B (NFκB) activation in hematopoietic progenitor cells (HPCs).51 Tumor Necrosis Factor alpha (TNFα)-mediated activation of IκB kinase (IKK) and NFκB was substantially reduced in the presence of VEGF. The inhibitory effect of VEGF on NFκB activation was shown to be independent of VEGFR-1 and VEGFR-2 since SU5416, a potent inhibitor of VEGFR-1 and VEGFR-2, did not prevent VEGF-induced inhibition of NFκB. NFκB plays an important role in B-cell, T-cell and dendritic cell development.52,53 The physiological consequences of VEGF-mediated NFκB inactivation in these cells has yet to be elucidated. Studies have shown that VEGF acts as an anti-apoptotic factor, protecting hematopoietic cells from programmed cell death.54 Both VEGF and VEGFR-2 are expressed in normal hematopoietic cells and in leukemic cells, and VEGF suppresses apoptosis induced by ionizing irradiation in both cell types.55 In addition, survival of CMK86 leukemia cells is increased by VEGF treatment in the presence of both etoposide and doxorubicin,56 with MCL1, a member of the Bcl-2 family of anti-apoptotic proteins, up-regulated in response to VEGF. Transfection of U937 myeloid leukemia cells with MCL1 decreased caspase 3 activity and increased cell viability in the presence of etoposide. When CMK86 cells were treated with VEGF, northern blot analysis revealed amplification of ZK7 mRNA.57 Transcription factor ZK7 is a member of the Krüppel family of genes 58 and is associated with the apoptotic-signalling pathway. ZK7 mRNA was increased in multiple leukemia cell lines and numerous normal tissues following VEGF treatment. U937 cells transfected with constitutively expressed ZK7 survived irradiation doses 3-4 times higher than nontransfected cells, and the number of apoptotic cells among ZK7-transfected U937 cells was reduced significantly.57 It is clear from these findings that VEGF protects hematopoietic cells from apoptosis and may directly contribute to tumorigenesis by prolonging the survival of leukemic cells, as well as indirectly via angiogenesis.

Evidence for VEGF Autocrine Signalling in Solid Tumors Not only is VEGF a major player in leukemias and lymphomas, it is also highly expressed in a variety of solid malignant tumors,59-61 and correlates with malignant disease progression.62,63 VEGF overexpression in tumors is associated with increased angiogenesis, proliferation and metastasis.64,65 A recently published study showed phosphorylated VEGFR-2 (KDR) expression in numerous solid tumors including three lung carcinomas, three breast carcinomas, three Non Hodgkin’s lymphomas, and one melanoma.66 Not only did endothelial cells stain positive for phosphorylated VEGFR-2 but many other cell types including macrophages, fibroblasts, and myofibrils. In all cases both neoplastic cells and surrounding stromal cells stained positive for phosphorylated VEGFR-2. The patterns of staining varied within and between sections with some cells displaying strong cytoplasmic staining and others having stronger nuclear staining. This may be explained by the internalization and redistribution of KDR once it is phosphorylated.67 Although similar patterns were observed in normal tissues staining was noticeably weaker than in tumors. Many other studies show that VEGFR-2 is expressed in a variety of nonmalignant tissues31,68,69 and tumors.70-72 Ovarian carcinoma cells were the first non-endothelial tumor cells shown to express VEGFR-2.2 Primary tumors, metastases of ovarian tumors, and cell lines derived from ovarian carcinomas were examined for VEGF and VEGF receptor expression. Three of four ovarian carcinoma cell lines expressed VEGF, VEGFR-1 and VEGFR-2 mRNA. VEGF protein was detected in the culture media from these cell lines. VEGF mRNA was elevated in all primary tumors and metastases, with immunoreactivity for VEGF localized to clusters of tumor cells and patches of stromal matrix, demonstrating VEGF production in primary ovarian tumors and metastases. VEGF was also released into the surrounding stromal matrix, where it is likely to contribute to tumor growth and metastasis in a paracrine manner through angiogenesis and increased vascular permeability. VEGFR-1 and VEGFR-2 were expressed on some tumor cells,

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and VEGFR-2 was expressed in primary tumor sites raising the possibility of an autocrine VEGF loop in ovarian cancer regulation. Pancreatic cancer is extremely aggressive with very poor prognosis.73 VEGF expression was demonstrated in pancreatic cancer,74,75 and both VEGF and VEGF receptor expression was elevated in pancreatic tumor cells in comparison to normal pancreatic tissue.3 VEGF colocalized with its receptors, VEGFR-1 and VEGFR-2, in many of the cancer cells. VEGF was detected in culture supernatants from all pancreatic cell lines tested.76 Coexpression of VEGF and its receptors in vivo and in vitro suggests a role for autocrine as well as paracrine stimulation of pancreatic tumor growth by VEGF. p44/42 MAPK was shown to mediate VEGF signalling via VEGFR-2, with c-fos transactivation as a possible downstream event.76 VEGF treatment increased DNA synthesis in Dan-G and AsPc-1 pancreatic cells. This effect was antagonized by an anti-VEGF neutralizing antibody and by soluble receptor fragments of VEGFR-1 and VEGFR-2. Transfection of truncated soluble forms of VEGFR-1 and VEGFR-2 disrupted the VEGF signalling pathway and significantly inhibited the growth of each cell type.76 VEGF did not stimulate VEGF-VEGFR signalling in normal pancreatic cells or in chronic pancreatitis, suggesting that this VEGF receptor system is a feature of transformed pancreatic cells and may contribute to the ‘angiogenic switch’ observed in the development of many tumors, promoting tumor progression.77 These observations provide evidence for the existence of a VEGF autocrine mitogenic loop in pancreatic cancer. In another malignancy, mesothelioma, which responds poorly to treatment, a VEGF autocrine loop appears to directly stimulate tumor cell growth.78 As with ovarian cancer and pancreatic cancer, malignant pleural mesothelioma (MM) cells produce VEGF and express VEGFR-1 and VEGFR-2.79 In four MM cell lines treated with recombinant VEGF, VEGFR-1 and VEGFR-2 phosphorylation was observed and cell proliferation increased.78 VEGF, VEGFR-1 and VEGFR-2 expression was also observed in MM biopsies.78,80 No correlation was observed between serum VEGF levels and vascular density within tumors,78,79 suggesting that in addition to its angiogenic activity, VEGF may promote tumor growth by direct pro-survival effects in tumor cells.

Autocrine VEGF Signalling in Breast Cancer VEGF expression in breast cancer is well documented and VEGF is produced by both macrophages and cancer cells in breast carcinoma.81,82 VEGF receptor expression has also been demonstrated on breast cancer cells.83,84 Dual staining for VEGF and its receptors on frozen sections from invasive breast carcinomas revealed their coexpression on breast cancer cells,84 suggesting that both paracrine and autocrine VEGF pathways play a role in breast cancer progression. VEGFR-1 and VEGFR-2 mRNA was identified in a number of breast cancer cell lines including T-47D, MCF7, MDA-MB-453, and MDA-MB-231, and these cancer cells were shown to respond to VEGF.85,86 VEGF stimulation increased the invasiveness of T-47D cells in the presence of fibronectin.85 ERK-1 and –2 were activated in response to VEGF in T-47D cells; PI3-K and its downstream target Akt were also activated. Since T-47D cells secrete VEGF87 it is likely that an autocrine VEGF loop exists within these cells. PI3-K activation has often been implicated in cellular invasion88 and may be the signalling mechanism in T-47D cells that mediates VEGF-induced cell invasion.

VEGF Stimulates Breast Cancer Invasion Anti-sense oligonucleotide inhibition of VEGF resulted in a sixty-five percent decrease in Matrigel invasion by MDA-MB-231 cells.89 Inhibition of invasion was reversed by the addition of recombinant VEGF demonstrating that cell invasion is a specific response to VEGF. Matrigel invasion by the highly invasive MDA-MB-435 cells was also inhibited by the anti-sense oligonucleotides. There are conflicting reports on VEGF receptor expression on the MDA-MB-231 and MDA-MB-435 cells but both cell lines have been shown to express NP-1 and VEGFR-1. Different results from studies of VEGF receptor expression may be due to

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differences in antibodies and techniques employed.85,89 VEGF-mediated invasion of these breast carcinoma cell lines was shown to be NP-1-dependent.89 Further investigations revealed that VEGF-mediated invasion was completely blocked by pertussis toxin, indicating that G-protein, Gαi, is essential to this pathway. The Gαi-coupled chemokine receptor, CXCR4, was previously shown to be expressed on breast cancer cells and to promote metastasis.90 CXCR4 expression on MDA-MB-231 and MDA-MB-435 cells was increased by VEGF, and anti-sense oligonucleotides to VEGF prevented cancer cell migration towards the CXCR4 ligand, SDF-1 (Stromal cell derived factor-1).89 Together this implies that CXCR4-dependent migration and invasion of these breast cancer cells requires VEGF. This was confirmed when a CXCR4-neutralizing peptide was shown to inhibit Matrigel invasion of the cells by seventy-five percent. Since the CXCR4 ligand, SDF-1, is found in tumor stroma and in tissues such as lymph and lung,90 the primary targets for breast cancer metastasis, VEGF-mediated CXCR4 expression may be important in breast cancer invasion and migration. Blockade of this pathway using anti-VEGF strategies and/or CXCR4-neutralizing peptides may help prevent tumor spread and metastasis in breast cancer patients.

VEGF Signalling in Tumor Cells Not only does VEGF have a direct influence on breast cancer invasion and migration, it has also been shown to act as a survival factor for metastatic breast carcinoma cells.86,91 Reduced VEGF expression induces apoptosis in these cells. Anti-sense VEGF oligonucleotides not only reduce VEGF expression by MDA-MB-231 and MDA-MB-435 cells but induce cell death, seen as a four-fold increase in annexin V staining (a marker for early apoptosis) and a three-fold increase in propidium iodide staining in transfected cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assays confirmed that this increased cell death was due to apoptosis.92 VEGF stimulation of NP-1 maintained elevated levels of PI3-K activity in MDA-MB-231 cells and inhibition of this PI3-K activity induced apoptosis.92 Studies in our own laboratory have shown that VEGF protects both human and murine breast carcinoma cells from apoptosis.86 Additional studies in our laboratory have identified NP-1 as the receptor involved in VEGF mediated protection of these breast cancer cells.93 Although VEGFR-2 mRNA was identified in 4T1 and MDA-MB-231 cells, the corresponding protein was not found. However, NP-1 receptor protein was expressed on both cancer cell lines. Both cell lines were treated with peptides which blocked NP-1 or VEGFR-2. Treatment of these cells with the anti-NP-1 peptide induced apoptosis but the anti-VEGFR-2 peptide had no effect. Confocal microscopy revealed that only the anti-NP-1 peptide bound to the cancer cells (Fig. 1). This evidence confirms a role for the NP-1 receptor in VEGF-mediated breast cancer cell protection from apoptosis. The α6β4 integrin is associated with progression of many solid tumors and with poor prognosis.94 Activation of α6β4 integrin promotes breast carcinoma cell survival.91,95 When MDA-MB-435 cells, which lack α6β4, were transfected with α6β4 their resistance to cell death increased. VEGF expression was also increased in these transfected cells, and their resistance to cell death was abrogated with the addition of VEGF anti-sense oligonucleotide. α6β4 was shown to increase VEGF translation through activation of the PI3-K/Akt pathway. Activation of this pathway induced phosphorylation of the transcriptional repressor, 4E-binding protein (4E-BP1), by mammalian target of rapamycin (mTOR), leaving it unable to associate with eukaryotic translation initiation factor 4E (eIF-4E). eIF-4E was then capable of initiating the translation of proteins such as VEGF. Transfection of the cells with eIF-4E anti-sense oligonucleotide significantly reduced the level of VEGF protein production in α6β4-transfected MDA-MB-435 cells. The PI3-K inhibitor, LY294002, and the mTOR inhibitor, rapamycin, had the same effect. Levels of apoptosis were greatly increased following these treatments, demonstrating the importance of the PI3K/mTOR pathway in regulating VEGF expression (Fig. 2). This study showed that the α6β4 integrin increased VEGF protein translation and thus protected breast carcinoma cells from apoptosis.

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Figure 1. Confocal Microscopy showing FITC-labelled anti-NP-1 peptide binding to MDA-MB-231 (A) and 4T1 (B) mammary adenocarcinoma cells, (original magnification x 400). Images are representative of a scan zoom of between 1 to 4.2-fold.

Anti-Angiogenic Therapy With the confirmation that VEGF plays a major role in angiogenesis within tumors a variety of anti-VEGF strategies to inhibit tumor growth and angiogenesis have been designed. Monoclonal antibodies (mAb) directed against VEGF have been tested in phase I trials.96,97 In phase II trials humanized anti-VEGF mAb stabilized breast cancer and induced remission in patients with relapsed metastatic breast carcinoma.98 Tyrosine kinase inhibitors to target the VEGF receptors have shown promise, and phase I trials have been initiated.99,100 Studies using ribozymes targeting VEGF mRNA have also been conducted and have entered clinical trials.101,102 The success of these anti-VEGF methodologies may be due not only to the inhibition of angiogenesis, but also to the inhibition of paracrine/autocrine VEGF signalling in the tumor cells themselves.2,84 It is likely that VEGF produced by both tumor cells and stromal cells within tumors binds to VEGF receptors on both the endothelial and neoplastic cells. In addition, there is substantial data in support of the presence of autocrine VEGF loops on tumor cells themselves,42,70,76 thus tumor cells are likely to mediate their own survival, invasiveness and migration through VEGF pathways.

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Figure 2. Diagram adapted from Bachelder RE, Ribick MJ, Marchetti A et al. p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB. J Cell Biol. 1999;147:1063-72.©Rockefeller University Press.

Development of anti-VEGF strategies to target angiogenesis and VEGF pathways in tumors should prove very effective in preventing the progression of many tumor types. However, with many other pro-angiogenic growth factors, such as basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGFβ) also elevated within tumors, blockade of VEGF alone is unlikely to prove universally effective. Chemotherapy and radiotherapy both increase VEGF expression13 and may therefore increase tumor resistance. Anti-VEGF treatments should block this therapy-induced resistance and VEGF-induced tumor cell survival, as well as inhibiting angiogenesis. Anti-VEGF treatments used in combination with conventional chemotherapy and radiotherapy should dramatically improve treatment for cancer patients, with some initial studies already providing promising results.13,103,104 Studies in our own laboratory have shown that combining anti-NP-1 peptide with taxotere or cisplatin increases the anti-tumor effects of these chemotherapeutic agents (Byrne AM, Bouchier-Hayes DJ, Harmey JH et al unpublished data).

Acknowledgements Funded under the Programme for Research in Third Level Institutions (PRTLI), administered by the HEA and Health Research Board Grant RP182/2000.

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80. Konig J, Tolnay E, Wiethege T et al. Coexpression of vascular endothelial growth factor and its receptor flt-1 in malignant pleural mesothelioma. Respiration 2000; 67:36-40. 81. Harmey JH, Dimitriadis E, Kay E et al. Regulation of macrophage production of vascular endothelial growth factor (VEGF) by hypoxia and transforming growth factor beta-1. Ann Surg Oncol 1998; 5:271-278. 82. Lewis JS, Landers RJ, Underwood JC et al. Expression of vascular endothelial growth factor by macrophages is up-regulated in poorly vascularized areas of breast carcinomas. J Pathol 2000; 192:150-158. 83. Speirs V, Atkin SL. Production of VEGF and expression of the VEGF receptors Flt-1 and KDR in primary cultures of epithelial and stromal cells derived from breast tumors. Br J Cancer 1999; 80:898-903. 84. de Jong JS, van Diest PJ, van der Valk P et al. Expression of growth factors, growth inhibiting factors, and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops. J Pathol 1998; 184:44-52. 85. Price DJ, Miralem T, Jiang S et al. Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells. Cell Growth Differ 2001; 12:129-135. 86. Pidgeon GP, Barr MP, Harmey JH et al. Vascular endothelial growth factor (VEGF) upregulates BCL-2 and inhibits apoptosis in human and murine mammary adenocarcinoma cells. Br J Cancer 2001; 85:273-278. 87. Yoshiji H, Gomez DE, Shibuya M et al. Expression of vascular endothelial growth factor, its receptor, and other angiogenic factors in human breast cancer. Cancer Res 1996; 56:2013-2016. 88. Adam L, Vadlamudi R, Kondapaka SB et al. Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol-3 kinase. J Biol Chem 1998; 273:28238-28246. 89. Bachelder RE, Wendt MA, Mercurio AM. Vascular endothelial growth factor promotes breast carcinoma invasion in an autocrine manner by regulating the chemokine receptor CXCR4. Cancer Res 2002; 62:7203-7206. 90. Muller A, Homey B, Soto H et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001; 410:50-56. 91. Chung J, Bachelder RE, Lipscomb EA et al. Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF translation: A survival mechanism for carcinoma cells. J Cell Biol 2002; 158:165-174. 92. Bachelder RE, Crago A, Chung J et al. Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res 2001; 61:5736-5740. 93. Barr MP, Duffy AM, Byrne AM et al. Neuropilin-1 receptor blockade using peptides, induced apoptosis of mammary adenocarcinoma cells. (Data Submitted) 94. Mercurio AM, Rabinovitz I. Towards a mechanistic understanding of tumor invasion-lessons from the alpha 6 beta 4 integrin. Semin Cancer Biol 2001; 11:129-141. 95. Bachelder RE, Ribick MJ, Marchetti A et al. p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB. J Cell Biol 1999; 147:1063-1072. 96. Gordon MS, Margolin K, Talpaz M et al. Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer. J Clin Oncol 2001; 19:843-850. 97. Margolin K, Gordon MS, Holmgren E et al. Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: Pharmacologic and long-term safety data. J Clin Oncol 2001; 19:851-856. 98. Sledge G, Miller K, Novotny W et al. A phase II trial of single-agent rhuMAb VEGF (recombinant humanized monoclonal antibody to vascular endothelial growth factor) in patients with relapsed metastatic breast cancer. Proc Am Soc Clin Oncol 2000; 19 (abstr 5c). 99. Smolich BD, Yuen HA, West KA et al. The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts. Blood 2001; 97:1413-1421. 100. Shaheen RM, Tseng WW, Davis DW et al. Tyrosine kinase inhibition of multiple angiogenic growth factor receptors improves survival in mice bearing colon cancer liver metastases by inhibition of endothelial cell survival mechanisms. Cancer Res 2001; 61:1464-1468. 101. Weng DE, Usman N. Angiozyme: A novel angiogenesis inhibitor. Curr Oncol Rep 2001; 3:141-146. 102. Sandberg JA, Parker VP, Blanchard KS et al. Pharmacokinetics and tolerability of an antiangiogenic ribozyme (ANGIOZYME) in healthy volunteers. J Clin Pharmacol 2000; 40:1462-1469. 103. Teicher BA, Holden SA, Ara G et al. Potentiation of cytotoxic cancer therapies by TNP-470 alone and with other anti-angiogenic agents. Int J Cancer 1994; 57:920-925. 104. Lee CG, Heijn M, di Tomaso E et al. Anti-Vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. Cancer Res 2000; 60:5565-5570.

CHAPTER 14

Vascular Endothelial Growth Factor C and Vascular Endothelial Growth Factor D: Biology, Functions and Role in Cancer Sarah E. Duff and Gordon C. Jayson

Abstract

V

ascular Endothelial Growth Factors (VEGF)-C and VEGF-D are angiogenic and lymphangiogenic members of the VEGF family. These two growth factors form a subgroup by virtue of their structural and functional similarities. VEGF-C and VEGF-D are ligands for VEGF receptors, VEGFR-2 and VEGFR-3 and signalling via VEGFR-3 is important for the development of the vascular system and maintenance and function of the lymphatic system. Differential cell surface antigen expression by blood vascular and lymphatic endothelial cells has provided a method of isolating lymphatic endothelial cells for further investigation of the roles of VEGF-C and VEGF-D. Novel tumor and lymphedema models have been constructed to analyze the in vivo functions of VEGF-C, VEGF-D and VEGFR-3. Although proliferating lymphatic endothelial cells have been identified in human tumors, the role of lymphatic vessel density and its relationship to the expression of lymphangiogenic growth factors has yet to be clarified fully. The expression of VEGF-C and VEGF-D in human tumors is associated with negative clinicopathological variables, but some conflicting results are found which may be explained by differences in methodology, tumor biology and microenvironmental influences. Evaluation of the therapeutic potential of manipulation of the VEGF-C/D signalling pathway requires further clarification of the functions of the growth factors in health and disease.

Introduction The vascular endothelial growth factor (VEGF) family is central to angiogenesis. VEGF-A (usually referred to as VEGF) is the most studied member of this family and its angiogenic role and that of its receptors is well described.1,2 Further members include placental growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E (reviewed by Partanen et al3 and Duff et al4). This chapter focusses on the angiogenic and lymphangiogenic members of the VEGF family, VEGF-C and VEGF-D, their molecular biology, functions and potential roles in malignancy.

Molecular Biology of VEGF-C and VEGF-D Appreciation of the importance of VEGF in angiogenesis and the lack of a known ligand for VEGFR-3 (Flt-4) led to the search for additional VEGF family members. VEGF-C was identified as a specific activator for VEGFR-3 isolated from conditioned medium of PC-3 human prostate cancer cells and was also found to stimulate VEGFR-2 by causing tyrosine autophosphorylation of the receptors.5 At the same time, Lee et al identified the identical VEGF and Cancer, edited by Judith H. Harmey. ©2004 Eurekah.com and Kluwer Academic / Plenum Publishing.

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growth factor from a cDNA library of the human glioma cell line G61, naming this protein vascular endothelial growth factor-related protein (VRP), now renamed VEGF-C.6 The mouse homologue of VEGF-D, c-fos-induced growth factor (FIGF), was identified by differential mRNA screening of fibroblasts from c-fos deficient and wild type mice.7 Human VEGF-D is 85% homologous to murine FIGF and was identified by computer-based homology searching for VEGF-related sequences and found to be a ligand for VEGFR-2 and VEGFR-3 in common with VEGF-C.8

VEGF-C and VEGF-D Genes The gene for murine VEGF-C maps to chromosome 8 and the equivalent human locus is chromosome 4q34.9 A high degree of conservation is seen in the organisation of the gene between mouse and man. The human gene is composed of more than 40 Kb of genomic DNA and comprises seven exons, separated by introns varying in length from 301 bp to over 10 Kb. The intron-exon boundaries are highly conserved and all the exons contain coding sequences.10 Exons 2 – 4 code for the VEGF-homology domain of the VEGF-C molecule. Transcription of the VEGF-C gene begins 523 bp upstream from the translation start site, resulting in a long 5′-untranslated region of mRNA. The promoter sequence contains putative binding sites for the transcription factors activator protein (AP) –2, specificity protein (Sp) -1, and nuclear factor kappaB (NF-κB) but no hypoxia-response elements (HREs).10 The lack of HREs in the gene promoter may explain why some authors have found that VEGF-C production, unlike VEGF, is unaffected by hypoxia.11,12 However, work on seminoma cell lines in culture, demonstrated upregulation of VEGF-C and VEGF-D gene expression in response to hypoxic culture conditions through unexplained mechanisms.13 The transcription factor NF-κB is induced by hypoxia and may contribute to hypoxia-induced upregulation of VEGF-C and D. The lack of a definite relationship between hypoxia and VEGF-C expression implies that VEGF-A and VEGF-C have distinct functions in the vascular system. NF-κB is also induced by various inflammatory stimuli and may account for the increase in VEGF-C production in response to inflammatory cytokines.12 Recently, insulin-like growth factor (IGF)-1 acting via the IGF-1 receptor (IGF-1R) has been shown to upregulate the expression of VEGF-C.14 Upregulation via the IGF-1/IGF-1R axis is likely to be mediated through the AP-2 and Sp-1 transcription factors and depends on intracellular signalling through phosphatidylinositol 3′-kinase (PI3-K) and also, to a lesser extent, mitogen-activated protein kinase (MAPK).14 Other factors stimulating the production of VEGF-C include platelet derived growth factor (PDGF), epidermal growth factor (EGF) (both of which also activate PI3-K signalling), transforming growth factor β (TGFβ), basic fibroblast growth factor (bFGF)15 and in a non-physiological setting, the tumor promotor phorbol myristate 12,13-acetate (PMA).11 The major human VEGF-C mRNA transcript is 2.4kb, although a 200-400 bp shorter minor mRNA species has been identified.5,10 The shorter species lacks the nucleotides of exon 4, corresponding to the VEGF-homology domain. Consequently, this transcript is unlikely to have VEGF-related biological function or relevance.10 The 3′ region of human VEGF-C mRNA contains a single AUUUA motif. This motif is implicated in the rapid turnover of many cytokine mRNAs and the presence of only a single motif may explain the greater stability of VEGF-C mRNA, compared with that of VEGF-A which contains multiple motifs. The gene for VEGF-D maps to human chromosome Xp22.16,17 The gene spans about 50 Kb and demonstrates similar organisation to the VEGF-C gene with 7 exons and 6 introns. The promoter region has no TATA box but contains an AP-1 binding site at –54 from the translation start site. This site contains consensus sequences for the c-jun/c-fos family of transcription factors, consistent with evidence that mouse FIGF expression is stimulated by c-fos expression.7 Preliminary studies demonstrate a role for hypoxia in upregulation of VEGF-D promoter activity in rat pulmonary microvascular smooth muscle cells. This hypoxia response element resides within a 523 bp fragment of the rat VEGF-D promoter.18

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Table 1. Comparison of molecular biological features of VEGF-C and VEGF-D VEGF-C

VEGF-D

Chromosome Gene

4q34 40 Kb, 7 exons AP-2, Sp-1, NK-κB binding sites

Xp22 50 Kb, 7 exons AP-1 binding sites

Upregulation

Inflammatory stimuli IGF, PDGF, EGF, TGF-β, FGF, serum, phorbol myrisate

c-fos expression Cell-cell contact mediated by cadherins, Possibly hypoxia

mRNA

2.4 Kb

2.3 and 2.8 Kb

Protein

Produced as preproprotein and proteolytically processed ~30% & 61% identity to VEGF-A and VEGF-D respectively in VEGF homology domain

Produced as preproprotein and proteolytically processed 31% & 61% identity to VEGF-A and VEGF-C respectively in VEGF homology domain

C-terminal extension, 37% identical to VEGF-D N-terminal extension, 25% identical to VEGF-D

C-terminal extension, 37% identical to VEGF-C N-terminal extension, 25% identical to VEGF-C

3 potential glycosylation sites, 2 of which are conserved with VEGF-D

3 potential glycosylation sites, 2 of which are conserved with VEGF-C

Open reading frame encodes a protein of 419 amino acids

Open reading frame encodes a protein of 354 amino acids

VEGFR-3 by all forms VEGFR-2 by proteolytically processed mature form

VEGFR-3 by all forms VEGFR-2 by proteolytically processed mature form

Receptor Binding

VEGF-D mRNA is found as a 2.3 kb transcript in most human tissues and is most abundant in heart, lung, skeletal muscle, colon and small intestine. The mRNA is also found as an additional less abundant 2.8 Kb transcript in skeletal muscle.8 Despite their similarities, VEGF-C and D expression are regulated differently. VEGF-D expression is not induced by cytokines but by activation of the proto-oncogene, c-fos 7 and VEGF-D mRNA expression is induced in mouse fibroblasts by cell-cell contact mediated by cadherin-11.19

VEGF-C and VEGF-D Proteins Within the VEGF family, VEGF-C and VEGF-D form a subgroup because of their structural and functional similarity and receptor specificity (Table 1, Fig. 1). Unlike VEGF-A which exists in a number of different isoforms due to alternative splicing of the mRNA, only a single protein is produced from VEGF-C or VEGF-D mRNA transcripts. However, both VEGF-C and VEGF-D are subject to extensive proteolytic processing resulting in proteins of varying molecular weight and functional ability according to the extent to which they have

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Figure 1. Schematic diagram representing the structure of the VEGF-C preproprotein. The VEGF-C protein consists of a signal sequence, N-terminal extension, VEGF homology domain and C-terminal extension. The cleavage sites are marked by arrowheads and the numbers of the flanking amino acid residues. The diagram beneath the VEGF-C structure shows the scale in amino acid residues. The 8 conserved cysteine residues in the VEGF homology domain are indicated by open circles.

been processed.20,21 The protein convertase family of enzymes, in particular furin, PC5 and PC7, are involved in VEGF-C processing.22 Overall, VEGF-D is 48% identical to VEGF-C.8 Structurally, the VEGF-C and VEGF-D subgroup is characterized by: a) Production as a pre-proprotein 20 b) Amino (N) and carboxy (C) terminal extensions. The C-terminal extension is unique in this subgroup of the VEGF family and rich in cysteine residues. The N- and C-terminal extensions are related in sequence between VEGF-C and –D, sharing 25% and 37% sequence identity, respectively.8 c) Progressive proteolytic cleavage of the terminal extensions to generate the mature forms of cytokine with increased receptor affinity.20,23 Proteolytic processing occurs both within and outside the secreting cell. Immature and partially proteolytically processed forms bind to VEGFR-3 (dissociation constant, Kd 130 pM) but full proteolytic cleavage is required for VEGFR-2 activating ability (Kd 410 pM).20,21 Mature VEGF-C consists of the 21 kD VEGF-homology domain, dimerized with non-covalent bonds, whereas the partially processed forms have a variety of molecular masses.20 The open reading frame of VEGF-C encodes a protein of 419 amino acids, whereas VEGF-D is 354 amino acids in length. d) Similarity to VEGF in the central part of the protein within the VEGF homology domain in which the cysteine knot structure typical to the family is conserved. Within the VEGF homology domain, VEGF-C is ~30% identical 5,6 and VEGF-D is 31% identical to VEGF.8 The two growth factors are 61% identical to one another within this region.8 e) Three potential N-linked glycosylation sites, 2 of which are conserved between VEGF-C and VEGF-D.6,8

VEGF Receptors The VEGF receptor family is a subgroup of the platelet derived growth factor (PDGF) receptor family. There are three main receptors, VEGFR-1 (Flt-1 or fms-like tyrosine kinase-1),

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VEGFR-2 (KDR or kinase domain-containing receptor/Flk-1 or fetal liver kinase-1) and VEGFR-3 (Flt-4) (reviewed in refs. 1,2,24). The VEGFR-3 gene maps to human chromosome 5q33-q35.25 The gene consists of 31 exons and differential mRNA splicing of exon 30 results in polypeptide isoforms that differ in length by 65 amino acid residues. This arises by alternative polyadenylation of mRNA and subsequent alternative splicing.26,27 The overall genomic organisation is similar to that of the VEGFR-1 and VEGFR-2 genes. The 5′ promoter region shows putative binding sites for transcription factors of the NF-1 (nuclear factor-1), AP-2, GATA (a group of factors containing a conserved zinc-finger DNA binding domain) and Ets (a group of factors that contain an ‘ets domain’ that interacts with the tetranucleotide GGAA) families and the 3′ promoter elements include Sp-1 sites and other areas that resemble the start sites found in genes that lack a TATA box.27 Inactivating mutations in the VEGFR-3 gene, including missense mutations and intragenic polymorphisms result in defective VEGFR-3 signalling and are described in families suffering from primary lymphedema.28,29 The VEGF receptors are typical of the PDGF family of receptor tyrosine kinases, with seven extracellular immunoglobulin homology domains and a split intracellular tyrosine kinase domain. VEGFR-3 is a highly glycosylated, relatively stable, cell surface associated kinase of approximately 180 kD. It is proteolytically cleaved in the fifth extracellular immunoglobulin-like domain and the resulting chains remain linked with disulfide bonds.30 Short and long isoforms of VEGFR-3 are recognized, differing in length in the C-terminus.26 The longer form predominates in human tissues and the two forms have different functional abilities as only activation of the long form is able to sustain fibroblast cell growth in soft agar cultures.31 Downstream signalling from VEGFR-3 is dependent on residues within the intracellular carboxy tail. Site directed mutagenesis of tyrosine residues in the C-terminal of the long form of VEGFR-3 abolishes signalling capacity.32 The sites of expression of VEGFR-3 differ from that of VEGFR-1 and VEGFR-2, which are predominantly expressed on vascular endothelial cells. VEGFR-3 is also expressed on vascular endothelium in early development but later becomes restricted mainly to the lymphatic endothelium33,34 and this fact may be used to define subsets of endothelial cells as lymphatic in origin.35 However, VEGFR-3 is not solely expressed on lymphatic endothelium in all situations. Expression is increased on the endothelial cells of angiogenic vessels in and around tumors36-39 and may also be seen in some fenestrated endothelia.40 However, VEGFR-3 does not seem to be upregulated on the neovasculature of the acute healing wound, although it is present at low levels on the vascular endothelium within chronic wound granulation tissue.41

VEGF-C and VEGF-D Signalling and Function In common with other members of the VEGF family, ligand binding is responsible for receptor dimerization, intracellular tyrosine kinase activity and autophosphorylation, leading to the activation of multiple intracellular signalling pathways.1 Proteolytic processing of the ligands and heterodimerization of VEGF family members influence the affinity of binding and outcome of receptor activation.1,20 VEGF-C and D are ligands for VEGFR-2 and VEGFR-3, but do not possess VEGFR-1 activating ability.5,8 VEGFR-3 signalling, through VEGF-C or D ligand binding, is crucial for the development of the vascular system and for maintenance and function of the lymphatic system. Its contribution to the vascular system is highlighted following disruption of VEGFR-3 signalling in embryonic mice, in which impaired remodelling of the primary vascular plexus and cardiovascular failure is seen at embryonic day (E) 9.5, but the development of primary vascular networks, vascular sprouting and endothelial cell differentiation is unaffected.42 Defective VEGFR-3 signalling results in primary lymphedema28,29 and murine lymphedema models have been created with inactivating mutations of VEGFR-3 43 or by using soluble VEGFR-3 molecules to inhibit VEGFR-3 signalling.44

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In Vitro Studies of VEGF-C and VEGF-D Function VEGF-C and VEGF-D activate both VEGFR-2 and VEGFR-3 and are consequently implicated in both angiogenic and lymphangiogenic pathways. Both VEGF-C and VEGF-D exhibit mitogenic effects for vascular and lymphatic endothelial cells and survival-promoting abilities for lymphatic endothelial cells through VEGFR-3.6,8,20,45-47 Angiogenic pathways, involving endothelial cell migration and activation, are stimulated by VEGF-C but with much less efficiency than by VEGF.5,20,48 VEGF-D also stimulates angiogenesis in three-dimensional culture systems.47 The study of lymphatic endothelial cells in culture has been hampered by the lack of available cells from the lymphatic microvasculature. Most studies have used endothelial cells from large lymphatic vessels such as the thoracic duct for analysis. These cells are unstable in culture, losing expression of known lymphatic antigens 49 and may not reflect the characteristics of the microvascular lymphatic endothelial cell in vivo. However, the origin of cell subtypes within cultured cell lines has been clarified which should provide a source of lymphatic endothelial cells for further definitive work. The human dermal microvascular endothelial cell (HDMEC) line has been shown to consist of a mixture of two separate, stable lineages of vascular and lymphatic endothelial cells that could be separated using antibodies to exploit their differential expression of the cell surface receptor VEGFR-3.45 Similar methodology exploiting the differential expression of podoplanin on lymphatic and blood vessel endothelial cells has been used to separate the two cell populations from dermal cell suspensions.35 Further modifications of this technique have been used to separate human dermal blood and lymphatic vascular endothelial cells by their differential expression of CD34 and CD31, whereby lymphatic endothelial cells were consistently CD34 negative/CD31 positive.50 Lymphatic endothelial cells obtained in this way and maintained in culture exhibited stable expression of the lymphatic specific cell markers over repeated passages, a finding which validates the use of these antigens for the detection of lymphatic vessels in tissues. Furthermore, the identification of differential expression of lineage-specific genes in blood vascular and lymphatic endothelial cells confirmed the close relationship between the two cell types, in that only relatively small differences were found between their expression profiles, although these differences provided insights into the specialized functions of the cells as well as pinpointing further molecular targets for investigation of vascular development, regulation of the immune response and lymphangiogenesis.50 The use of these cell separation techniques and further investigation of the gene groups that are expressed differently between blood and vascular endothelial cells will be helpful for clarification of the precise role and functions of VEGF-C, VEGF-D and VEGFR-3.

In Vivo Studies of VEGF-C and VEGF-D Function The role of VEGF-C and D in angiogenic and lymphangiogenic pathways has been investigated in a variety of animal models. Fully processed VEGF-C has VEGF-like actions, increasing blood vessel permeability in guinea pig skin in a dose-dependent manner20 and inducing angiogenesis in ischaemic tissues.51 In the corneal neovascularization assay, both VEGF-C and D are capable of inducing angiogenesis 47,48 The lymphangiogenic potential of VEGF-C has been examined in the differentiated chorioallantoic membrane (CAM) assay.52 Lymphatic vessels were identified by various methods including in situ hybridization with probes for VEGFR-2 and VEGFR-3. The application of recombinant VEGF-C to the CAM induced lymphangiogenesis but not angiogenesis and caused proliferation of lymphatic endothelial cells as detected by bromodeoxyuridine labelling.52 The roles of VEGF-C, VEGF-D and VEGFR-3 in dermal lymphatic development have been demonstrated using transgenic mouse models. These models use the keratin promoter K14 to control expression of VEGF-C or VEGFR-3 transgenes and localize their expression to the basal layer of the epidermis. An increase in K14-driven transgene expression is seen from

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E14.5. Jeltsch et al showed that transgenic mice producing VEGF-C in the skin, manifest dermal lymphatic vessel enlargement and lymphatic endothelial cell proliferation without alteration in blood vasculature.53 Similar effects have been shown using transgenic VEGF-D and a mutant form of VEGF-C possessing VEGFR-3 activating ability only.46 Conversely, transgenic mice producing the soluble fusion protein VEGFR-3-Immunoglobulin (VEGFR-3-lg), in the skin, demonstrate inhibition of lymphatic development.44 Dermal lymphatics developed in early fetal life but regressed after E14.5 and were absent by birth. The transgenic mice were characterized by swollen feet at birth and older mice showed thickened dermal and subcutaneous layers of the skin and a complete lack of dermal lymphatics but again, no effect was seen on the development of the vascular system.44 Combination of these two models to produce a double transgenic mouse expressing both mutant VEGF-C or VEGF-D in the skin and soluble VEGFR-3-Ig fusion protein showed inhibition of lymphatic hyperplasia although transgene expression remained high.46 In summary, dermal lymphangiogenesis is stimulated by VEGF-C and VEGF-D and mediated via VEGFR-3.46 Additional animal models of lymphoedema have been created by inactivating mutations of VEGFR-3 in the germline 28,43 and will be useful in the study of therapeutic interventions for this condition. Novel animal models of lymphedema have been constructed in the rabbit ear,54,55 the mouse tail55,56 and the regenerating lizard tail.57 These models will provide an exciting arena in which to investigate the actions of the lymphangiogenic cytokines in greater detail. Using new animal models, Yoon et al have confirmed that VEGF-C naked plasmid DNA is capable of ameliorating lymphedema and has a lymphangiogenic effect.55 Tumor models have been constructed in which over-expression of VEGF-C or VEGF-D by the tumor is demonstrated eg. in melanoma,58 breast cancer,59-62 pancreatic islet cell tumors63 and gastric tumors.64 Results of these studies show increased aggressiveness of the transfected cancer cell lines, intra-tumoral lymphangiogenesis, dilated and increased numbers of peri-tumoral lymphatics, enhanced rates of lymph node metastasis and increased tumor angiogenesis. In some studies, these effects have been abrogated by antibodies directed against VEGFR-360 or VEGF-D.61 Although VEGF-C and VEGF-D cause developmental lymphangiogenesis acting through VEGFR-3, whether lymphangiogenesis occurs within spontaneously arising tumors has been questioned. The presence of lymphatics within VEGF-C over-expressing animal tumor models has been demonstrated immunohistochemically in xenograft studies.59-61 In a transgenic model, mice who express VEGF-C driven by the rat insulin promoter (Rip) targeted to the β-cells of the endocrine pancreas were crossed with Rip1Tag2 mice who develop non-lymphangiogenic, non-metastatic pancreatic β-cell tumors. The consequent VEGF-C over-expressing tumors developed lymphatics that were located around and in the peripheries of the developing tumor.63 It is possible that in xenograft models, the injection of malignant cell suspensions to create tumors entraps pre-existing lymphatics within the developing tumor mass, whereas transgenic animal models may more accurately reflect the situation in spontaneously arising human tumors.65 The question of whether immunohistochemically detected intra-tumoral lymphatics are functional is the subject of current debate.66 Previously, functional lymphatics have only been found within 100 µm of the invading edge of murine sarcoma xenografts, increased interstitial pressure within the tumor contributing to the lack of functioning lymphatics within the tumor mass.67 More recently, Padera et al demonstrated a lack of viable intra-tumoral lymphatics and the presence of functional peri-tumoral lymphatics in mouse xenografts, which showed clearly that peripheral peri-tumoral lymphatics are sufficient to enable lymphatic metastasis.68 This mirrors the situation in the transgenic mouse pancreatic tumor model, in which enlarged peri-tumoral lymphatics, but not intra-tumoral lymphatics, were responsible for increased lymph node metastasis and lymphatic invasion.63

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Ex Vivo and Human Studies of VEGF-C and VEGF-D Function From the in vitro and animal studies, it is clear that VEGF-C induces hyperplasia of pre-existing lymphatic vessels,53 induces the development of new lymphatics, 52 acts as a survival factor for lymphatic endothelial cells 45,46 and influences angiogenesis.48 However, the precise involvement of VEGF-C, VEGF-D, VEGFR-3 and lymphangiogenesis in human malignancy is not fully understood.69,70 Investigation of the lymphatic vasculature has been limited by the lack of specific lymphatic vessel markers, but this situation has changed dramatically with the discovery and use of more specific antibodies and antibody combinations.71-73 Antibodies of great interest include those directed against lymphatic vessel hyaluronan receptor (LYVE)-1,74 Prox-1 (a homeobox gene product involved in early lymphatic development)75 and podoplanin.76 The antibodies described against these antigens largely demonstrate specific lymphatic vessel staining and are the subject of current intense investigation. Lymphatic metastasis is an important determinant of prognosis and the most common pathway of tumor dissemination for many solid malignancies. Although lymphangiogenesis has been demonstrated in VEGF-C over-expressing animal tumor models, until recently only correlational evidence existed in human tumors and whether lymphangiogenesis occurred within human tumors was unclear (reviewed in refs. 66,77-79). In human cutaneous melanoma, lymphatics, but not lymphangiogenesis, were observed at the tumor margin; the peripheral lymphatics were pre-existing and trapped within the expanding tumor mass.80 However, recent studies have refuted the lack of lymphangiogenesis in melanoma.81,82 Dadras et al82 studied a retrospective series of metastatic and non-metastatic melanomas and demonstrated proliferating intra- and peri-tumoral lymphatic vessels. Furthermore, the number of intra-tumoral lymphatics, peri-tumoral lymphatics, average lymphatic vessel size and lymphatic vessel area was higher in metastatic melanomas than in matched non-metastastic tumors. Further evidence for tumor-derived lymphangiogenesis is derived from work by Beasley et al83 These authors demonstrated the existence of proliferating lymphatics within head and neck cancer stroma and an association between intra-tumoral lymph vessel density (LVD) and neck node metastases.83 These studies have raised the possibility of intra- or peri-tumoral LVD as a potential prognostic marker, analogous to blood vessel microvessel density (MVD), to predict patients at risk of poor outcome. However, the presence of proliferating lymphatic endothelial cells within a tumor may not necessarily equate with functional lymphatic channels and no functional assessment has been possible in these studies. Different tumor types may differ with respect to the relative importance of lymphangiogenesis as a means of lymphatic metastasis. Recent work has shown a lack of proliferating lymphatics in and around breast cancer, despite active angiogenesis, and a low LVD in comparison to blood vessel microvascular density.84 Despite these findings, breast cancer was still able to spread to the regional lymph nodes through pre-existing peri-tumoral lymphatics.84 In general, these recent studies have demonstrated the presence of lymphatic vessels within some human cancers and that the lymphatic endothelial cells in these vessels appear to be proliferating. The studies quoted above have all employed antibodies against LYVE-1 as a specific lymphatic marker. It is not clear whether findings will be generalized to the other specific lymphatic markers, such as Prox-1 or podoplanin, nor what the clinical relevance of these findings will be in the context of human malignancies unstudied so far, particularly those with a predominantly lymphatic route of spread. Further research is required to confirm these results in different tumor types, to investigate the relationship of LVD and lymphangiogenesis to tumor expressed lymphangiogenic growth factors and determine the best antibody or antibody combination to allow comparison between centres and studies.

VEGF-C and VEGF-D in Human Malignancy The expression of VEGF-C and VEGF-D is increased in a variety of human malignancies.70,85 Positive associations have been found between the expression of VEGF-C and VEGF-D in malignant tissue with adverse clinicopathological features (Tables 2 and 3). Multiple tumor

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Table 2. Expression of VEGF-C in human tumors

Tumor Type reference

Methods

Breast carcinoma 86 Breast carcinoma 88 Breast carcinoma 87

IHC IHC IHC, RT-PCR RPA, IHC RT-PCR IHC IHC RT-PCR RPA, RTPCR, IHC IHC RTQ-RTPCR IHC RT-PCR, IHC RT-PCR, IHC IHC IHC RT-PCR, IHC IHC IHC RT-PCR, ISH, IHC IHC IHC IHC RT-PCR, IHC RT-PCR, WB RT-PCR

Breast carcinoma 90 Breast carcinoma 89 Cervical carcinoma 112 Cervical carcinoma 110 Cervical carcinoma 111 Colorectal carcinoma 102 Colorectal carcinoma 99,103 Colorectal carcinoma 98 Colorectal carcinoma 104 Colorectal carcinoma 100 Colorectal carcinoma 101 Endometrial carcinoma 107 Esophageal SCC 105 Esophageal SCC 106 Gallbladder carcinoma 139 Early gastric carcinoma 96 Gastric carcinoma 97 Gastric carcinoma 94 Gastric carcinoma 93 Early gastric carcinoma 95 Gastric carcinoma 91 HNSCC 120 Lung adenocarcinoma 119 Melanoma 82 Mesothelioma 140 Neuroblastoma 141 NSCLC 118 NSCLC 130 NSCLC 117 NSCLC 116

ISH, IHC RT-PCR RTQ-RTPCR IHC IHC IHC IHC

Clinicopathological Associations of Increased Tumor VEGF-C Expression with Regard to: Lymphatic Venous Lymph Prognosis Invasion Invasion Node Metastasis NA NA NS

p=0.0131 NS NS

p=0.0165* NS p=0.0356**

NA NA p=0.036 NS

NA NA

NS p=0.04 NS p=0.002 p=0.006 NS

NA NA NA p=0.0232§ NS NA

p<0.01 p=0.006

p<0.01 NS

p<0.01 p=0.023

p=0.0322§ NA

NS p=0.0032

NS NS

NS p=0.0025

NA NS trend

NA

NA

NS

NA

p<0.0001 NS p<0.01

p=0.0006 NS p<0.01

p=0.0008 p=0.01§ NS, p=0.085 NS p<0.01 NA

p<0.001 p=0.01 p<0.05

NS NS NS

p<0.001 p=0.09 p<0.05

p<0.01* NA NA

p<0.05 p=0.04 p=0.02 p<0.05

NS NS, p=0.07 NS p<0.01

p<0.05 NS NS p<0.05

p<0.01* NS trend NA p<0.011§

NA

NA

p<0.0001

NS trend

NA NA p=0.004 NS NA

NS, p=0.053 NA NA NA NA

NA NA NA

Tended to NA be more likely NS NA NS NS NS NA

p<0.05 NA p=0.011 NA

NA NA NS NA

p<0.01 NS p=0.021 p<0.0001

p<0.001§ p=0.003* p<0.05* NS

continued on next page

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154

Table 2. Continued

Tumor Type reference

Methods

Oral SCC 121 Oral SCC 122 Ovarian carcinoma 125 Pancreatic carcinoma 109 Prostate carcinoma 108 Papillary thyroid carcinoma 115 Papillary thyroid carcinoma 142 Thyroid carcinoma 113

IHC IHC IHC IHC ISH RT-PCR

Clinicopathological Associations of Increased Tumor VEGF-C Expression with Regard to: Lymphatic Venous Lymph Prognosis Invasion Invasion Node Metastasis NA NA p<0.01 NA NA

NA NS NA NA NA NA

p=0.008 p=0.003 p=0.008 p<0.01 p=0.0083 p<0.05

NS p<0.05* p=0.046* NS NA NS

IHC

NA

NA

p=0.0191

NS

RT-PCR, IHC

NA

NA

p<0.0001

NA

HNSCC, head and neck squamous cell cancer; IHC, immunohistochemistry; ISH, in situ hybridisation; NA, not assessed; NS not specified; NSCLC, non-small cell lung carcinoma; RPA, ribonuclease protection assay; RTQ-RT-PCR, real-time quantitative reverse-transcriptase polymerase chain reaction; RT-PCR, reverse-transcriptase polymerase chain reaction; SCC, squamous cell carcinoma; WB, Western blot. § p-value on multivariate analysis, showing that increased VEGF-C expression was an independent prognostic indicator *p-value on univariate analysis, that failed to show significance on multivariate analysis **p-value on univariate analysis, no multivariate analysis performed.

types have been investigated with respect to VEGF-C expression, these include breast,86-90 gastric,91-97 colorectal,98-104 oesophageal,105,106 endometrial,107 prostate,108 pancreas,109 cervical,110-112 thyroid,113-115 non-small cell lung cancers,116-118 lung adenocarcinoma,119 head and neck 120 and oral squamous carcinomas121,122 and laryngeal cancers.123 The expression of VEGF-C in human tumors tends to be associated with lymphatic invasion, lymph node metastasis and poorer survival (Table 2). However, this does not hold true for all tumors studied nor for all clinicopathological features assessed. The relationship with outcome is particularly variable; some studies find VEGF-C expression to be an independent prognostic indicator, whereas others show a significant effect on univariate analysis which loses significance in multivariate assessment (Table 2). The level of association of growth factor expression with the various clinicopathological variables examined also differs between studies and tumor types. With regard to tumor VEGF-D expression, the observed associations with clinicopathological factors are even more variable than those seen with VEGF-C (Table 3). In lung adenocarcinoma, a negative relationship was seen between VEGF-D mRNA expression and lymphatic invasion,119 leading the authors to propose that a balance between the VEGF family members, in particular VEGF-C and VEGF-D, is important in directing tumor behaviour, a view that has been repeated by George et al in the context of colorectal tumorigenesis.101 Conversely, however, an independent prognostic effect and a positive association between increased VEGF-D expression and lymph node metastasis has been illustrated in breast,124 ovarian125 and colorectal cancer.126

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Table 3. Expression of VEGF-D in human tumors

Tumor Type reference

Breast carcinoma 124 Breast carcinoma 88 Breast carcinoma 89 Colorectal carcinoma 143 Colorectal carcinoma 102 Colorectal carcinoma 126 Colorectal carcinoma 101 Endometrial carcinoma 144 Early gastric carcinoma 96 HNSCC 120 Lung adenocarcinoma 119

Clinicopathological Associations of Increased Tumor VEGF-D Expression with Regard to: Methods Lymphatic Venous Lymph Prognosis Invasion Invasion Node Metastasis IHC IHC RT-PCR RT-PCR, WB, IHC RPA, RTPCR, IHC IHC RT-PCR, IHC IHC IHC RT-PCR, WB RT-PCR

Ovarian carcinoma125 IHC Papillary thyroid carcinoma 115 RT-PCR

NA NA NA NS

NA NA NA NS NS

NA NA

p=0.01 NA Lower VEGF-D with LI, p=0.006 NA NA

NA NA p<0.0001 NS NA

p=0.0238 NS NS p<0.01

p=0.045§ NS NA NS, p=0.15

NA

NA

p=0.02 NS

p=0.026§ NA

p=0.0004 p=0.03 NS

p=0.017* NA NA

NA

Tended to be NA less likely

NA NA

p=0.02 NS

p=0.004§ NS

HNSCC, head and neck squamous cell cancer; IHC, immunohistochemistry; NA, not assessed; NS not specified; RPA, ribonuclease protection assay; RT-PCR, reverse-transcriptase polymerase chain reaction; WB, Western blot; LI, lymphatic invasion § p-value on multivariate analysis, showing that increased VEGF-D expression was an independent prognostic indicator *p-value on univariate analysis, that failed to show significance on multivariate analysis

Several possible explanations can be invoked to account for the conflicting outcomes and conclusions of the studies: these relate to methodological areas, tumor biology and the tumor microenvironment. Part of the differences observed may be understood by the use of numerous different methodologies and consequent difficulties in extrapolation of results and comparison between studies.4 For example, studies assessing tumor mRNA levels cannot be directly equated with those examining expression of tissue protein as mRNA expression does not necessarily correlate with protein expression. Tumor biology will be responsible for some of the discrepancies seen. In colorectal cancer, Furodoi et al99 demonstrated that the association of VEGF-C expression with negative clinicopathological factors was only present on assessment at the invading tumor edge and not at the central or superficial parts of the tumor. This makes biological sense, as the invading edge is the site of the host-tumor interface and the most active part of the tumor. Assessment of growth factor expression at this location may provide more clinically relevant information than either global or random tumor assessment. As discussed earlier, it is possible that different tumor types may rely to varying levels on recruitment of pre-existing lymphatic vessels in the peri-tumoral area or on lymphangiogenesis (either in the peri-tumoral area or within the

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tumor) to facilitate lymphatic spread. The relative levels of reliance on these different methods may in turn influence the associations seen between growth factor expression and clinicopathological factors. Far from being a passive process, tumor lymphatic invasion and lymph node metastasis may be directed by paracrine pathways. Tumor expression of chemokine receptors has been noted,127-129 which allows chemotaxis towards ligand-secreting tissues, such as lymphatic endothelial cells,35 potentially increasing and aiding tumor cell entry into lymphatic channels. The expression of different VEGFRs by tumor cells has also been identified in varying tumor types 130-136 which may influence tumor cell behaviour by autocrine (and paracrine) growth enhancement and potentially increase tumor cell survival, via the action of tumor (and stromal cell) secreted ligands. So, the site of growth factor expression, the balance between levels of different tumor-produced growth factors and their biological activity and the expression of receptors on malignant cells results in an extremely complex situation which differs within each tumor and complicates the interpretation of studies. The microenvironment influences tumor behaviour and the differing compositions of individual tumor microenvironments may provide a further key to the varying conclusions of human tumor studies. The level of inflammatory response differs greatly and can contribute to upregulated VEGF-C expression,11,12 the extent of growth factor proteolytic processing (hence altering growth factor biological activity) and enhanced angiogenic and lymphangiogenic responses.137 Tumor associated macrophages secrete lymphangiogenic growth factors 112 which may also enhance peri-tumoral lymphangiogenesis and lymphatic invasion. The concurrent expression of other growth factors in the tumor milieu may also influence the activity of VEGF-C.15 In summary, although tumor expression of VEGF-C and VEGF-D is increased in various human tumors and generally associated with negative clinicopathological factors, the relationship between growth factor expression, intra- and peri-tumoral LVD, MVD, lymphatic and venous invasion, lymph node metastasis and survival, is far from clear. Interpretation of studies is hampered by differences in methodology, tumor biology and microenvironment, which are difficult to define and quantify and likely to confound the results observed.

Potential Therapeutic Roles While the precise roles of VEGF-C and VEGF-D in malignancy remain to be further clarified and elucidated, it is known that both cytokines are angiogenic and lymphangiogenic during development and in the adult. In the context of cancer, new therapeutic approaches include anti-angiogenic and anti-lymphangiogenic strategies, which could exploit alteration of signalling via the VEGF-C/VEGF-D/VEGFR-2/3 pathways. This principle is illustrated by the use of monoclonal antibodies directed against VEGF-D, blocking VEGFR-2 and VEGFR-3 interaction and producing reduced angiogenesis, lymphangiogenesis and lymphatic spread in a mouse tumor model.138 Potential routes to inhibit the lymphangiogenic pathways include neutralising antibodies to VEGF-C, VEGF-D138 or VEGFR-3,15 soluble extracellular receptor domains to sequester VEGF-C/VEGF-D in the extracellular environment,44 tyrosine kinase inhibitors and inhibitors of downstream signalling molecules. Alternatively, enhancement of lymphangiogenic signalling might provide a treatment option for patients with secondary lymphedema that occurs following the treatment of malignant disease, e.g., arm lymphedema in breast cancer patients, following radiotherapy or surgery. This could theoretically be treated by targeting lymphangiogenic growth factor expression to areas of lymphatic loss.54,55 Unfortunately, studies increasing or decreasing the effects of VEGF-C, VEGF-D and/or signalling via VEGFR-2 or VEGFR-3 may have unwanted side effects such as lymphedema or enhanced lymphatic metastasis in tumors. Consequently, greater understanding of the function and actions of these growth factors are required before the clinical use of effective treatments for the management of both benign and malignant disease.

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Conclusions VEGF-C and VEGF-D possess angiogenic and lymphangiogenic properties and act via VEGFR-2 and VEGFR-3. Both in vitro and animal studies illustrate their important role in the progression of malignancy. Although studies of the lymphatic vasculature in human malignancy have been limited by lack of specific markers, recent work shows that lymphatics are present in some tumors, although peri-tumoral lymphatics may be the most important route for metastatic spread. The expression of VEGF-C and VEGF-D is increased in many human malignancies. Future work with specific lymphatic markers will clarify the role of pre-existing lymphatic vessel recruitment versus new lymphatic development in the lymphatic spread of tumors, define the relative importance of these pathways to lymphatic metastasis in different tumors and further elucidate the relationship between the lymphangiogenic growth factors and clinicopathological variables. The role of LVD as a prognostic marker and its relationship to VEGF-C and VEGF-D expression in different tumors requires further investigation. Encouraging results from the inhibition and enhancement of lymphangiogenic signalling pathways show potential for their development for future therapeutic roles in the treatment of cancer.

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Index A

C

α-interferon, see Interferon-α Acidic fibroblast growth factor (aFGF) see Growth factors Acute lymphoblastic leukemia (ALL) see Leukemia Acute myeloid leukemia (AML) see Leukemia Adenocarcinoma (AC) 29, 44, 54-59, 64, 107, 108, 110-112, 138, 153-155 Adenomatous polyposis coli (APC) 65 Age-related macular degeneration (AMD) 6, 7, 19 Akt 2, 4, 21, 22, 134, 137, 138, see also Protein kinase B Angiogenesis 1, 3-7, 12, 17, 19-23, 26-32, 34, 35, 40-44, 48-51, 54-58, 60, 64-66, 68, 69, 72-77, 79, 83, 84, 87-98, 107-113, 117, 118, 122-124, 126-130, 133, 135, 136, 139, 140, 145, 150-152, 156 Angiogenesis inhibitor 27, 65, 79, 95 Anti-angiogenesis 21, 30, 44, 48, 51, 66, 68, 69, 72, 74, 79, 83, 86, 91, 96, 98, 107, 108, 111-113, 129, 139, 156 Antisense 6, 15, 29, 44, 49, 50, 60, 77, 97, 111, 137, 138 Apoptosis 2, 22, 26, 27, 29-35, 44, 57, 67, 76, 83, 87, 89, 92, 107, 111, 112, 133-136, 138 Astrocytoma 72-74 Autocrine 2, 3, 20, 27, 50, 83, 84, 86-90, 93, 95, 97, 98, 133-137, 139, 156

C6 glioma 72, 75, 76 Cancer 1, 6, 7, 19, 20, 22, 23, 26, 31-35, 40-44, 49, 54, 56-60, 64-69, 73, 79, 84, 86, 96, 97, 107-112, 117, 125, 127, 128, 137-140, 145, 151, 152, 154-157 breast 26, 31, 33, 40-44, 137-139, 151, 152, 156 colorectal 1, 6, 32, 42, 64-69, 154, 155 esophageal 54, 57, 58, 60 pancreatic 107, 110-112, 137 Cartilage 5, 135 Caspase 22, 30, 31, 33, 136, 140 Celecoxib (Celebrex) 64 Central nervous system (CNS) 72-75, 76, 134 Chronic lymphocytic leukemia (CLL) see Leukemia Chronic myeloid leukemia (CML) see Leukemia Chronic myelomonocytic leukemia (CMML) see Leukemia Chronic myeloproliferative disorder 84 c-Jun N-terminal kinase (JUNK) 21, 22 Circulating endothelial cells (CEC) 84, 96 Circulating VEGF 32, 42, 43, 56, 57, 92, 96, 97 Clinical trials 68, 69, 79, 95, 139 Colorectal cancer see Cancer Colorectal cancer metastasis 67 Coronary artery disease (CAD) 17, 19 Cyclooxygenase 2 (COX-2) 44, 64-67, 91, 96 COX-2 inhibitor 44, 65, 67, 96

B Barrett’s disease 54-56, 59 Basic fibroblast growth factor (bFGF) see Growth factors Bcl-2 2, 22, 26-35, 89, 92, 136 Bcr-abl 91, 97 Bevacizumab 68, 69, 96 Bone 2, 3, 5, 7, 43, 57, 83, 85, 86, 88, 90, 91, 94, 95, 133, 135 Bone marrow angiogenesis (BMA) 83, 86, 88, 90-94, 96, 98 Breast cancer see Cancer

D Differentiation 1, 5, 7, 19, 21, 26, 42, 59, 73, 84, 85, 89, 98, 117, 122-124, 128, 130, 149

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E Endochondral bone formation 5, 7 Endothelial cells (EC) 1-4, 19-23, 26, 27, 29-31, 34, 35, 41, 42, 44, 48-50, 55-57, 59, 65, 67-69, 74-77, 79, 83-85, 86-90, 93, 95-98, 107-112, 117, 118, 122-124, 126, 128-130, 133, 135, 136, 145, 149-152, 156 Endothelial cell apoptosis 31, 67, 76, 111 Endothelium 3, 19, 20, 23, 43, 49, 50, 59, 67, 72, 74, 75, 79, 84, 86, 123, 149 Ependymomas 72 Epidermal growth factor (EGF) see Growth factors Esophageal cancer see Cancer Essential thrombocythemia (ET) 87, 90, 91 Extracellular regulated kinase (ERK1/2) 21, 22

Granulocyte macrophage colony stimulating factor (GM-CSF) 49, 85, 87, 90, 97 Growth factors acidic fibroblast (aFGF) 1, 40, 41 basic fibroblast (bFGF) 1, 7, 27, 40, 41, 44, 48-51, 56, 75, 76, 88, 90-96, 107-109, 111, 129, 140, 146, see also Fibroblast growth factor 2 epidermal (EGF) 3, 20, 30, 40, 41, 44, 58, 68, 74-76, 108, 122, 146, 147 hepatocyte (HGF) 1, 4, 41, 48, 90-92, 95, 96 keratinocyte (KGF) 3, 20 placental (PlGF) 1, 3, 4, 20, 22, 48, 145 platelet derived (PDGF) 3, 20, 41, 67, 74-79, 90, 108, 146-149 TGF-α and β see Transforming growth factors α and β VEGF see Vascular endothelial growth factors

F 5-fluorouracil (5-FU) 6, 57, 58, 68, 69 Familial adenomatous polyposis (FAP) 64 Farnesyl transferase inhibitor (FTI) 83, 96 Fetal liver kinase 1 (Flk-1) 3, 4, 20, 29, 42, 49, 67, 74, 77, 84, 109-111, 134, 149, see also Kinase insert domain-containing receptor and VEGFR-2 Fibrin 40, 41, 86, 117-119, 124, 126-130 Fibrin E-fragment (FnE) 119, 121, 122, 124-126, 128-130 Fibrinogen (Fgn) 117-120, 122-124, 126, 128-130 Fibroblast growth factor 2 (FGF-2) 20, see also Basic fibroblast growth factor FK-binding-protein (FKBP12) 69 Flk-1/KDR (Fetal liver kinase 1/kinase insert domain-containing receptor) 3, 20, 74, 77, 109, 110 Fms-like tyrosine kinase (Flt-1) 3, 20, 29, 42, 50, 67, 74, 84, 89, 97, 109, 110, 134, 135, 148, see also VEGFR-1 Focal adhesion kinase (FAK) 21, 22

G Gene expression 2, 17, 20, 22, 28, 55, 56, 59, 146, 150, 151 Glioblastoma multiforme 72-76 Glioma 22, 72-79, 146

H Hematopoietic malignancy 86, 87 Hemostasis 117 Hepatocyte growth factor (HGF) see Growth factors Hodgkin’s disease (HD) 93, 94, 136 Human dermal microvascular endothelial cell (HDMEC) 30, 120-123, 125-129, 150 Human umbilical endothelial cell (HUVEC) 4, 30, 31, 123, 135 HuR 12, 15, 16 HuR nucleocytoplasmic shuttling sequence (HNS) 15 Hypoxia 2, 3, 12-17, 20, 27, 28, 32, 34, 48, 56, 57, 60, 65, 75, 76, 93, 110, 134, 146, 147 Hypoxia-inducible factor 1 (HIF-1) 2, 3, 1214, 16, 17, 20, 28, 56, 65, 76

I Interferon-α (IFNα) 92, 96, 98 Interleukin-3 (IL-3) 49 Interleukin-8 (IL-8) 1, 30, 4851, 91, 108, 109, 111, 112 Immunomodulatory drug (ImiDs) 96, 98 Inducible nitric oxide synthase (iNOS) 22 Insulin-like growth factor-1 ( (IGF-1) 3, 146 IGF-1R 146

165

Index Intercellular adhesion molecule-1 (ICAM-1) 122, 129 Internal ribosome entry sites (IRES) 15, 16 Intra-ocular neovascularization 6 Iressa 44, 68 Irinotecan 6, 68, 69

J JUNK see c-Jun N-terminal kinase

K Kaposi’s sarcoma 30, 110 Keratinocyte 3, 20, 50, 51, 109, 133 Keratinocyte growth factor (KGF) see Growth factors Kinase insert domain-containing receptor (KDR) 3, 4, 20, 29, 42, 49, 59, 67, 74, 77, 84, 109, 110, 111, 112, 134, 136, 149, see also Fetal liver kinase 1 and VEGFR-2

L Leucovorin 6, 68, 69 Leukemia acute lymphoblastic (ALL) 83, 84, 88-90, 104, 135 acute myeloid (AML) 33, 83, 84, 87-90, 97, 98 chronic lymphocytic (CLL) 83, 84, 92, 93 chronic myeloid (CML) 83, 84, 90-92, 96 chronic myelomonocytic (CMML) 87, 90 Lymphangiogenesis 1, 43, 50, 59, 84, 150-152, 155, 156 Lymphatic system 145, 149 Lymphoma 26, 83, 84, 87, 89, 93, 94, 97, 136

M Macrophages 2, 20, 49, 56, 57, 59, 65, 66, 74, 78, 84, 85, 93, 94, 124, 133, 134, 136, 137, 156 Mast cells 50, 91, 93 Mastocytosis 90, 91 Melanocyte 50 Melanoma 20, 26-29, 42, 48-51, 109, 110, 136, 151-153

Metastasis 19, 26, 28, 34, 40, 41, 43, 44, 48, 49, 55, 66-68, 107-113, 117, 136, 138, 151-157 Microenvironment 3, 28, 34, 84-86, 89, 95-97, 109, 129, 130, 145, 155, 156 Microvascular 30, 34, 50, 56, 77, 78, 86, 91, 117, 122, 146, 150, 152 Microvessel density (MVD) 31, 32, 41, 42, 49, 55-58, 60, 66, 87-90, 92-94, 96, 111, 112, 152, 156 Migration 19-23, 26, 27, 34, 48, 55, 65, 77, 86-88, 92, 95-97, 108, 109, 117, 120-125, 127-130, 134, 138, 139, 150 Mitogen-activated protein kinase (MAPK) 21, 22, 29, 76, 137, 146 Monoclonal gammopathy 94 mRNA (messenger RNA) 2, 3, 5, 6, 12-17, 20, 22, 28, 49, 50, 55, 56, 59, 60, 67, 68, 72, 74-76, 87-89, 91-95, 109-111, 134-139, 146, 147, 149, 154, 155 mRNA stability 13, 14, 15, 17 mTOR 67, 68, 69, 138 mTOR inhibitor 68, 69, 138 Multiple myeloma (MM) 83, 84, 87, 94-98, 135, 137 Myelodysplastic syndrome (MDS) 83, 84, 87-90, 96, 98 Myelofibrosis with myeloid metaplasia (MMM) 83, 87, 90-92, 96, 97 Myeloproliferative disorder (MPD) 84, 87, 90, 91, 92, 96, 97

N Neovascularization 6, 19, 27, 30, 31, 41, 49, 67, 74, 93, 97, 108, 133, 135, 150 Neuropilin-1 (NP-1) 4, 29, 84, 90, 97, 134, 135, 137, 138, 140 Nuclear transcription factor kappa B (NF-κB) 28, 29, 136, 146 Nitric oxide (NO) 19-23, 66 Non-endothelial cell 1, 133 Non-Hodgkin’s lymphoma (NHL) 84, 93, 94

O Oligodendrogliomas 72 Ovarian angiogenesis 5 Oxygen tension 2, 13

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P

S

p53 28, 33, 44, 58, 65, 66, 76, 140 Pancreatic cancer see Cancer Paracrine 3, 4, 20, 56, 83, 84, 86, 87, 89, 90, 93, 95, 97, 98, 133-137, 139, 156 Placental growth factor (PlGF) see Growth factors Plasma cell (PC) 94, 95, 145 Plasmin 2, 76, 118, 119, 123, 126, 128-130 Platelet derived growth factor (PDGF) see Growth factors Postthrombocythemic myeloid metaplasia (PTMM) 91 Preclinical model 72, 77, 79, 96 Pro-angiogenic molecule 54, 60, 67, 95, 96, 107, 109 Prognosis 31-33, 40, 41, 43, 49, 51, 54, 55, 57, 64, 66, 73, 74, 78, 83, 88, 93-95, 97, 137, 138, 152-155 Prognostic factor 31, 66, 68, 88, 89, 92, 94 Prognostic value 31, 49, 56, 58, 59, 66, 84, 91 Proliferation 3-5, 12, 19-23, 26, 27, 29, 31, 44, 48, 50, 55, 58, 67-69, 72, 74, 78, 83, 84, 86-89, 91, 93-97, 108, 109, 117, 120-125, 128-130, 134-137, 150, 151 Protein kinase B (PKB) 134, 140, see also Akt Protein kinase C (PKC) 21, 22 PTK787 97, 107, 111, 112

Severe combined immunodeficient (SCID) 30, 89-91, 93, 96, 97 Severe combined immunodeficient (SCID) mice 30, 90, 93, 97 Skeletal growth 1, 5 Squamous cell carcinoma (SCC) 51, 54, 55, 57-60, 153, 154

R Radiation resistance 79 Rapamycin 67-69, 138, see also mTOR Ras 3, 4, 22, 65, 66, 76, 96 Receptor tyrosine kinase inhibitor (RTKI) 69, 83, 97, 111 Retinopathy 6, 12, 17 Ribonuclease 154, 155

T Targeted therapy 79 Thalidomide 83, 96, 98 Thrombin 86, 118, 119, 128-130 Thrombospondin-1 30, 31 Tumor necrosis factor α (TNF-α) 1, 29, 30, 41, 48, 56, 88, 90, 92, 108, 136 Transcriptional activation 13, 14, 17 Transforming growth factor α (TGF-α) 1, 3, 41, 56, 108 Transforming growth factor-β (TGF-β) 1, 3, 20, 27, 41, 48, 57, 108, 109, 140, 146, 147 Transforming growth factor β-1 (TGFβ-1) 20, 27, 57 Tumor growth 1, 12, 16, 19, 26, 30, 41-44, 49, 55, 60, 67, 69, 72, 77-79, 83, 84, 86, 90, 93, 96, 112, 113, 136, 137, 139 Tumor progression 26, 29, 33, 48, 58, 65, 66, 74, 93, 137 Tyrosine kinase 1, 3, 4, 20, 21, 42, 44, 51, 59, 67-69, 74, 77-79, 83, 84, 95, 97, 107, 109-112, 134, 139, 148, 149, 156 Tyrosine kinase inhibitor 44, 51, 67-69, 77-79, 83, 97, 107, 111, 139, 156

U Ubiquitin proteosome pathway 12

Index

V Vascular endothelial growth factor (VEGF) 1-7, 12-17, 19-23, 26-32, 34, 35, 40-44, 48-51, 54-60, 64-69, 72-79, 83-98, 107-113, 117, 118, 120-130, 133-140, 145-157 Circulating 32, 42, 43, 56, 57, 92, 96, 97 C (VEGF-C) 1, 20, 43, 50, 54, 59, 68, 84, 89, 94, 97, 145-157 D (VEGF-D) 1, 20, 43, 50, 59, 68, 84, 97, 145-152, 154-157 pathway 21, 68, 69, 137, 139, 140 receptor (VEGFR) 1, 3-7, 19-23, 42, 43, 49-51, 56, 59, 67-69, 74, 77-79, 83-93, 95-98, 134-138, 145-152, 156, 157 receptor-1 (VEGFR-1) 1, 3-5, 7, 19-23, 42, 50, 67, 68, 74, 77, 78, 84, 85, 87, 89, 91, 93, 96, 97, 134-137, 148, 149, see also Flt-1 receptor-2 (VEGFR-2) 1, 3, 4, 6, 19-23, 29, 42, 43, 49-51, 56, 59, 67-69, 74, 77-79, 84, 85, 87, 89-93, 97, 98, 134-138, 145-150, 156, 157, see also Flk-1 and KDR Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) 40, 108-111 Vascular permeability factor (VPF) 2, 40-42, 79, 108-111, 117, 133 VEGF/VPF receptors 109, 110 Von Hippel-Lindau (VHL) 2, 3, 6, 14, 16, 17, 76

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