<%BANNER%>

Regulation of Breast Cancer Cell Growth and Survival by the Interaction of Focal Adhesion Kinase and Vascular Endothelia...

Permanent Link: http://ufdc.ufl.edu/UFE0022461/00001

Material Information

Title: Regulation of Breast Cancer Cell Growth and Survival by the Interaction of Focal Adhesion Kinase and Vascular Endothelial Growth Factor Receptor-3
Physical Description: 1 online resource (103 p.)
Language: english
Creator: Hunt, Darrell
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: breast, cancer, kinases, vegfr
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Vascular endothelial growth factor receptor-3 (VEGFR-3, flt-4) is a receptor tyrosine that is overexpressed in a variety of human carcinomas, but its role in carcinogenesis is not fully elucidated. Here, we characterized the effects of VEGFR-3 overexpression in human breast cancer cells using two model systems. First, we chose a cell line with low endogenous VEGFR-3 expression, MCF7, and constructed stable transfectants overexpressing VEGFR-3 called MCF7-VEGFR-3. In parallel, we used siRNA to downregulate VEGFR-3 expression in a cell line with high endogenous VEGFR-3 expression, BT474. VEGFR-3 overexpression increased cellular proliferation, motility and promoted cell survival and anchorage-independent growth in MCF7 cells. All of these cellular characteristics were inhibited by the downregulation of VEGFR-3 in BT474 cells. Importantly, VEGFR-3 overexpression increased tumor formation, evidenced by a tumor incidence of 93% in MCF7-VEGFR-3 xenografts compared to no tumors formed by MCF7-pcDNA3 xenografts in the absence of hormonal stimulation. With estrogen stimulation, MCF7-VEGFR-3 xenografts were ten times larger in volume than MCF7-pcDNA3 xenografts. FAK is a tyrosine kinase important for cell signaling and a VEGFR-3 binding partner. We developed a novel anticancer small molecule targeting the FAK-VEGFR-3 protein-protein interaction. We used molecular docking and in silico screening of a small molecule library and identified C4 (Chlorpyramine hydrochloride), a small molecule that is capable of mimicking the peptide that binds to FAK. Co-crystalization of FAK with C4 revealed that binding of C4 occurred at the VEGFR-3 binding site. As predicted, C4 decreases colocalization of FAK and VEGFR-3. In vitro C4 causes dephosphorylation of FAK and VEGFR-3, resulting in inhibition of breast cancer cell proliferation, and subsequent induction of apoptosis at 48 hours. In vivo, C4 reduced tumor growth in the BT474 and MCF7-VEGFR-3 models by 80% (all P values < 0.01) compared with vehicle treated controls. Concomitant administration of C4 with doxorubicin had a great antitumor effect and gave 85% reduction in both tumor models. Our data demonstrate that small molecule inhibitors of FAK binding sites can be identified as lead compounds to provide the basis for novel specific cancer therapeutic agents.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Darrell Hunt.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cance, William G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022461:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022461/00001

Material Information

Title: Regulation of Breast Cancer Cell Growth and Survival by the Interaction of Focal Adhesion Kinase and Vascular Endothelial Growth Factor Receptor-3
Physical Description: 1 online resource (103 p.)
Language: english
Creator: Hunt, Darrell
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: breast, cancer, kinases, vegfr
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Vascular endothelial growth factor receptor-3 (VEGFR-3, flt-4) is a receptor tyrosine that is overexpressed in a variety of human carcinomas, but its role in carcinogenesis is not fully elucidated. Here, we characterized the effects of VEGFR-3 overexpression in human breast cancer cells using two model systems. First, we chose a cell line with low endogenous VEGFR-3 expression, MCF7, and constructed stable transfectants overexpressing VEGFR-3 called MCF7-VEGFR-3. In parallel, we used siRNA to downregulate VEGFR-3 expression in a cell line with high endogenous VEGFR-3 expression, BT474. VEGFR-3 overexpression increased cellular proliferation, motility and promoted cell survival and anchorage-independent growth in MCF7 cells. All of these cellular characteristics were inhibited by the downregulation of VEGFR-3 in BT474 cells. Importantly, VEGFR-3 overexpression increased tumor formation, evidenced by a tumor incidence of 93% in MCF7-VEGFR-3 xenografts compared to no tumors formed by MCF7-pcDNA3 xenografts in the absence of hormonal stimulation. With estrogen stimulation, MCF7-VEGFR-3 xenografts were ten times larger in volume than MCF7-pcDNA3 xenografts. FAK is a tyrosine kinase important for cell signaling and a VEGFR-3 binding partner. We developed a novel anticancer small molecule targeting the FAK-VEGFR-3 protein-protein interaction. We used molecular docking and in silico screening of a small molecule library and identified C4 (Chlorpyramine hydrochloride), a small molecule that is capable of mimicking the peptide that binds to FAK. Co-crystalization of FAK with C4 revealed that binding of C4 occurred at the VEGFR-3 binding site. As predicted, C4 decreases colocalization of FAK and VEGFR-3. In vitro C4 causes dephosphorylation of FAK and VEGFR-3, resulting in inhibition of breast cancer cell proliferation, and subsequent induction of apoptosis at 48 hours. In vivo, C4 reduced tumor growth in the BT474 and MCF7-VEGFR-3 models by 80% (all P values < 0.01) compared with vehicle treated controls. Concomitant administration of C4 with doxorubicin had a great antitumor effect and gave 85% reduction in both tumor models. Our data demonstrate that small molecule inhibitors of FAK binding sites can be identified as lead compounds to provide the basis for novel specific cancer therapeutic agents.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Darrell Hunt.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cance, William G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022461:00001


This item has the following downloads:


Full Text

PAGE 1

REGULATION OF BREAST CANCER CELL GROWTH AND SURVIVAL BY THE INTERACTION OF FOCAL ADHESION KINASE AND VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-3 By DARRELL LAMONT HUNT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

PAGE 2

2008 Darrell Lamont Hunt 2

PAGE 3

In Memory of John Wesley Hunt Sons carry the dreams of their fathers. 3

PAGE 4

ACKNOWLEDGMENTS No product of this magnitude comes to fruition without the hard work of a lot of people. I say thank you to every one who helped me fulfill this dream, but there are a few people that I need to address individually. Priscilla McAucliffe, thanks for expanding my imagination. I would not have dreamed of getting a second doctoral degree if you had not opened the door. William Cance, my mentor, thanks for the opportunity to train at the University of Florida, for your unflinching support (even when things didnt stick to the wall), and for giving me the space to grow as a basic scientist. Elena Kurenova and Vita Golubovskaya, there isnt a word in the English language that can adequately express my gratitude for your daily guidance, your help shaping my ideas and your keen insight into problem-solving. My family, my friends and my wife, we are one step closer. 4

PAGE 5

TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 TABLE OF CONTENTS .................................................................................................................5 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................10 CHAPTER 1 INTRODUCTION AND BACKGROUND...........................................................................12 Focal Adhesion Kinase...........................................................................................................12 Identification, Binding Partners and Signal Transduction..............................................12 FAK Transduces Survival Signals...................................................................................13 FAK Promotes Motility...................................................................................................14 FAK and Breast Cancer...................................................................................................15 In vitro......................................................................................................................15 Murine......................................................................................................................16 Human......................................................................................................................16 VEGFR-3................................................................................................................................17 Initial Characterization, Binding Partners and Signal Transduction...............................17 Ligands............................................................................................................................19 VEGFR-3 is a Key Mediator of Lymphangiogenesis.....................................................20 In vitro......................................................................................................................20 Murine......................................................................................................................20 Human......................................................................................................................21 VEGFR-3 Functions in Blood Vessel Formation............................................................21 VEGFR-3 and Breast Cancer..........................................................................................22 In vitro......................................................................................................................22 Murine......................................................................................................................22 Human......................................................................................................................23 VEGFR-3 Ligands and Breast Cancer............................................................................24 VEGFR-3 and Tumor-associated Angiogenesis.............................................................25 FAK and VEGFR-3 Bind in Breast Cancer Cells...........................................................25 5

PAGE 6

2 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-3 PROMOTES BREAST CANCER CELL PROLIFERATION, MOTILITY, AND SURVIVAL IN VITRO AND TUMOR FORMATION IN VIVO....................................................................27 Abstract...................................................................................................................................27 Introduction.............................................................................................................................28 Materials and Methods...........................................................................................................29 Cell lines..........................................................................................................................29 Antibodies and Reagents.................................................................................................30 Western Blot....................................................................................................................30 Transfection.....................................................................................................................31 BrdU Incorporation Assay...............................................................................................31 Immunocytochemistry.....................................................................................................32 MTT (Cell Viability) Assay............................................................................................33 Motility and Invasiveness Assay using Matrigel Modified Boyden Chamber................33 RT-PCR...........................................................................................................................33 Colony Formation Assay.................................................................................................34 Animal Model..................................................................................................................34 Statistical Analysis..........................................................................................................35 Results.....................................................................................................................................35 Establishment of Model Systems to Examine the Effects of VEGFR-3 Overexpression in Breast Cancer.................................................................................35 VEGFR-3 Increases Breast Cancer Cell Viability and Proliferation..............................37 Overexpression of VEGFR-3 Increases Breast Cancer Cell Motility and Invasion.......37 VEGFR-3 Promotes Survival in Breast Cancer Cells Under Stress Conditions.............38 VEGFR-3 Promotes Anchorage-independent Growth....................................................39 VEGFR-3 Promotes Growth of Breast Tumors in an Animal Model.............................39 Discussion...............................................................................................................................41 3 NOVEL FOCAL ADHESION KINASE INHIBITOR C4 TARGETED TO THE BINDING SITE OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3 INHIBITS BREAST CANCER PROLIFERATION IN VITRO AND IN VIVO................51 Abstract...................................................................................................................................51 Introduction.............................................................................................................................52 Material and Methods.............................................................................................................55 Virtual Screening.............................................................................................................55 Co-Crystallization of C4 and Focal Adhesion Kinase FAT domain...............................56 X-Ray Data Collection and Refinement..........................................................................57 Cell lines..........................................................................................................................57 Antibodies and Reagents.................................................................................................58 Immunocytochemistry.....................................................................................................58 Assays of Cell Viability...................................................................................................59 Western Blot Analysis.....................................................................................................60 BrdU Incorporation Assay...............................................................................................61 Motility and Invasion Assay............................................................................................62 6

PAGE 7

RT-PCR...........................................................................................................................62 Animal Model..................................................................................................................63 Statistical Analysis..........................................................................................................63 Results.....................................................................................................................................64 Site Selection and Computational Docking for High Throughout Virtual Screening of Drug-like Compounds to Develop a Specific VEGFR-3-FAK Inhibitor................64 C4 Decreases Viability of Many Cancer Cell Types at Low Micromolar Concentrations.............................................................................................................65 Crystal Structure of Cocrystalized C4 and FAT..............................................................65 C4 Specifically Decreases Viability of Breast Cancer Cells and Sensitize Tumor Cells to Chemotherapy.................................................................................................65 C4 Treatment Affects Colocalization and Leads to Redistribution of FAK-VEGFR3 Complexes....................................................................................................................67 C4 Causes Dose-dependent Dephosphorylation of VEGFR-3 and Decreases Total Phosphorylation of FAK..............................................................................................68 C4 Causes Timeand Dose-dependent Decreases in Proliferation of VEGFR-3 Expressing Cells...........................................................................................................69 C4 Treatment Leads to Apoptosis...................................................................................69 C4 Specifically Decreases Motility and Invasion of Breast Cancer Cells......................70 C4 Suppresses Tumor Growth In Vvivo..........................................................................71 Discussion...............................................................................................................................72 LIST OF REFENCES....................................................................................................................96 BIOGRAPHICAL SKETCH.......................................................................................................103 7

PAGE 8

LIST OF TABLES Table page 3-1 X-ray data collection and refinement statistics..................................................................95 8

PAGE 9

LIST OF FIGURES Figure page 2-1 Establishment of model systems .......................................................................................44 2-2 VEGFR-3 increases viability and proliferation of breast cancer cells..............................45 2-3 VEGFR-3 increases motility of breast cancer cells...........................................................46 2-4 VEGFR-3 promotes resistance to apoptosis......................................................................47 2-5 VEGFR-3 promotes anchorage-independent growth of breast cancer cells......................48 2-6 VEGFR-3 increases tumor formation and tumor growth in xenotransplant mouse model. ...............................................................................................................................49 3-1 Site selection for high throughout virtual screening of drug-like compounds to develop small molecule FAK inhibitors............................................................................77 3-3 Fluorescence immunostaining of endogenous FAK, paxillin, and VEGFR-3 in BT474 cells and scatter plot analysis of colocalization. ..................................................80 3-4 C4 treatment causes dose-dependent dephosphorylation of VEGFR-3 and FAK. MCF7-VEGFR-3...............................................................................................................82 3-6 C4 specifically decreases motility and invasion of breast cancer cells in a dose-dependent manner..............................................................................................................86 3-7 C4 reduces tumor growth in xenotransplant mouse model................................................87 3-10 C4 treatment changes localization of endogenous FAK and VEGFR-3 in BT474 cells, but does not affect distribution of paxillin................................................................93 9

PAGE 10

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REGULATION OF BREAST CANCER CELL GROWTH AND SURVIVAL BY THE INTERACTION OF FOCAL ADHESION KINASE AND VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-3 By Darrell Lamont Hunt December 2008 Chair: William Cance Major: Medical SciencesMolecular Cell Biology Vascular endothelial growth factor receptor-3 (VEGFR-3, flt-4) is a receptor tyrosine that is overexpressed in a variety of human carcinomas, but its role in carcinogenesis is not fully elucidated. Here, we characterized the effects of VEGFR-3 overexpression in human breast cancer cells using two model systems. First, we chose a cell line with low endogenous VEGFR-3 expression, MCF7, and constructed stable transfectants overexpressing VEGFR-3 called MCF7-VEGFR-3. In parallel, we used siRNA to downregulate VEGFR-3 expression in a cell line with high endogenous VEGFR-3 expression, BT474. VEGFR-3 overexpression increased cellular proliferation, motility and promoted cell survival and anchorage-independent growth in MCF7 cells. All of these cellular characteristics were inhibited by the downregulation of VEGFR-3 in BT474 cells. Importantly, VEGFR-3 overexpression increased tumor formation, evidenced by a tumor incidence of 93% in MCF7-VEGFR-3 xenografts compared to no tumors formed by MCF7-pcDNA3 xenografts in the absence of hormonal stimulation. With estrogen stimulation, MCF7-VEGFR-3 xenografts were ten times larger in volume than MCF7-pcDNA3 xenografts. 10

PAGE 11

FAK is a tyrosine kinase important for cell signaling and a VEGFR-3 binding partner. We developed a novel anticancer small molecule targeting the FAK-VEGFR-3 protein-protein interaction. We used molecular docking and in silico screening of a small molecule library and identified C4 (Chlorpyramine hydrochloride), a small molecule that is capable of mimicking the peptide that binds to FAK. Co-crystalization of FAK with C4 revealed that binding of C4 occurred at the VEGFR-3 binding site. As predicted, C4 decreases colocalization of FAK and VEGFR-3. In vitro C4 causes dephosphorylation of FAK and VEGFR-3, resulting in inhibition of breast cancer cell proliferation, and subsequent induction of apoptosis at 48 hours. In vivo, C4 reduced tumor growth in the BT474 and MCF7-VEGFR-3 models by 80% (all P values <0.01) compared with vehicle treated controls. Concomitant administration of C4 with doxorubicin had a great antitumor effect and gave 85% reduction in both tumor models. Our data demonstrate that small molecule inhibitors of FAK binding sites can be identified as lead compounds to provide the basis for novel specific cancer therapeutic agents. 11

PAGE 12

CHAPTER 1 INTRODUCTION AND BACKGROUND Breast cancer is the most common form of non-cutaneous cancer in women, affecting one in every nine women in the United States of America. An estimated 250,230 new cases of in situ and invasive breast cancer will be diagnosed and approximately 41,000 breast-cancer related deaths are expected to occur in 2008. The National Cancer Institutes estimates that approximately 2.6 million women with a history of breast cancer were alive in 2007. The resulting financial burden to American society in treatment costs alone exceeds 8 billion dollars per year [1]. The emotional burden on families is incalculable. Like most invasive cancers, malignant breast cells must acquire six hallmarks as described by Hanahan and Weinberg: 1) autonomy to exogenous mitogenic growth signals, 2) insensitivity to antigrowth signals, 3) resistance toward apoptosis, 4) limitless replicative potential, 5) sustained angiogenesis, and 6) the ability to invade tissue and metastasize [2]. Most of these functions are mediated by increases in the gene products of proto-oncogenes, many of which are tyrosine kinases. This work focuses on how the interaction of two tyrosine kinases, focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3), regulates breast cancer cell growth and survival. Focal Adhesion Kinase Identification, Binding Partners and Signal Transduction Focal adhesion kinase (FAK) is a 125 kDA non-receptor tyrosine kinase that localizes to focal adhesions, which are sites of integrin-clustering where cells make contact with the extracellular matrix. FAK was originally discovered as a substrate for tyrosine phosphorylation in v-src transformed chicken embryonic fibroblasts [3, 4] and consists of 1063 amino acids with a central kinase domain flanked by large aminoand carboxy-termini [3]. 12

PAGE 13

The amino-terminus contains the FERM (band 4.1, ezrin, radixin, and moesin) homology domain and mediates FAKs interaction with a diverse group of proteins including receptor tyrosine kinases (epidermal growth factor receptor, platelet-derived growth factor receptor), apoptosis related proteins (p53, Receptor-Interacting Protein), and, potentially, the integrin-1 subunit [3, 5-7]. The amino-terminus also contains the site of autophosphorylation, tyrosine 397. Phosphorylation of this tyrosine allows for efficient binding of p60 src via its SH2 domain to form a FAK-src complex [8]. The FAK-src complex further enhances FAKs kinase activity through the phosphorylation of FAK at tyrosines 576 and 577. Other SH2-interacting proteins also bind to this critical site of autophosphorylation, including the p85 subunit of PI-3 kinase leading the activation of AKT, Grb7, and Shc connecting FAK to the Grb2-SOS pathway of ERK 1/2 activation [9]. The carboxy-terminus is a key regulator of FAK function. It contains the focal adhesion targeting (FAT) domain, the protein sequence responsible for the subcellular localization of FAK to focal adhesions, which is critical for FAK function [10, 11]. The carboxy-terminus of FAK (FAK-CD) serves as the binding site for a large variety of proteins including paxillin at the focal adhesion targeting domain, p130 CAS (CAS) at the proximal proline-rich SH3 domain, Grb2 at tyrosine 925, GTPase-activating protein Graf, and p190GEF, a Rho-A guanine nucleotide exchange factor. FAK Transduces Survival Signals FAK is required for survival signal transduction promoted by ligation of integrins by extracellular matrix proteins [12]. The phosphorylation and kinase activity of FAK are upregulated by integrin clustering or the binding of integrins to the extracellular matrix, resulting in the formation of macromolecular complexes of FAK with other non-receptor kinases, adaptor molecules, and cytoskeletal proteins [13-15]. These multi-protein complexes activate both the 13

PAGE 14

Ras-MAPK and PI-3 kinase signaling pathways, resulting in the upregulation of ERK 1/2 and AKT phosphorylation [12, 14, 15]. Overexpression of constitutively activated FAK anchored to the cell membrane promoted cell survival of Madin-Darby canine kidney cells, specifically constitutively activated FAK promoted resistance to anoikis, apoptosis induced by detachment of a cell from the extracellular matrix [16]. In addition to its effects on survival, constitutively activated FAK also transformed this cell line resulting in anchorage-independent growth, and, subsequent increased tumorigenicity [16]. Consistent with these results, Hungerford, et. al., demonstrated that inhibition of FAK by microjection of an antibody targeted to the FAT domain of FAK resulted in significant increases in apoptosis of newly attached chick embryonic fibroblasts [17]. FAK also promoted resistance to apoptosis induced by serum deprivation of mouse embryonic stem cells [18]. This effect of FAK was shown to be related to its ability to suppress p53-mediated apoptosis [18]. FAK Promotes Motility In addition to its pro-survival effects, multiple studies have demonstrated that FAK is required for integrinand growth factor-stimulated cell migration through its ability to form macromolecular complexes with (1) src family kinases and PI-3 kinase at its autophosphorylation site tyrosine 397, (2) p130Cas at its proximal carboxy-terminal proline-rich SH3 domain, and (3) activated receptor tyrosine kinases through its amino-terminal FERM domain [19-24]. These multi-protein complexes regulate cellular changes important for motility including actin stress fiber formation, focal adhesion turnover, and crk-mediated Rac-1 activation [25, 26]. In further support of FAKs role in promoting cellular motility studies demonstrate that FAK inhibition by endogenous FAK kinase inhibitor FIP200, overexpression of 14

PAGE 15

FAK-CD, dephosphorylation of FAK by PTEN, and small molecule targeting of FAKs ATP-binding site significantly reduced cell migration [21, 27-29]. FAK is necessary for completion of murine gestation [25]. FAK deficient murine embryos exhibited general mesodermal defects, characterized by abnormalities of notochord and somite formation and growth retardation of the heart and vasculature, resulting in death at E8.5 [25]. Fibroblasts from these FAK deficient embryos exhibited decreased cell motility, indicating that part of the mesodermal pathology may be due to failure of appropriate cell migration [25]. FAK and Breast Cancer In vitro In agreement with its function in non-cancerous cells, FAK overexpression promotes cell survival of breast cancer cells. Inhibition of FAK expression using antisense oligonucleotides resulted in detachment and apoptosis of breast cancer cells [30]. These results were further corroborated by Xu, et. al, demonstrating that displacement of FAK from focal adhesions by plasmid or adenoviral-mediated overexpression of the carboxy-terminal domain of FAK (FAK-CD) resulted in FAK dephosphorylation and degradation with subsequent rounding, detachment and apoptosis of breast cancer cells [31, 32] The pro-survival effects of FAK are partially mediated by FAKs protein-protein interactions. FAK binds to and sequesters the death-domain receptor protein receptor-interacting protein (RIP), inhibiting its pro-apoptotic binding with death-receptor complexes [7]. FAK also binds to, sequesters, and promotes the degradation of p53, inhibiting its pro-apoptotic functions [33]. FAK is also a primary mediator of breast cancer cell motility, connecting to macromolecular complexes and inducing signal transduction pathways known to be necessary for cell migration and invasion. For instance, FAK directly binds to and phosphorylates the guanine nucleotide exchange factor p190GEF, augmenting its ability to activate the small G15

PAGE 16

protein Rho A [34]. RhoA has been shown to increase breast cancer cell motility through NF-KB mediated upregulation of urokinase plasminogen activator expression [35]. Binding urokinase plasminogen activator to its cognate receptor activates a signal transduction leading to increased phosphorylation of ERK 1/2 in breast cancer cells [36, 37]. ERK 1/2 promotes cell motility through its activation of myosin light chain kinase [38]. Murine Using a transgenic mouse model with mammary gland specific interruption of FAK expression, Lahlou, et. al. demonstrated that FAK expression is required for the transition of premalignant breast hyperplasias to breast carcinoma induced by mouse mammary tumor virus [39]. Suppporting the role of FAK in breast cancer motility and survival, downregulation of FAK expression using FAK-specific shRNA or decreased FAK function using FAK-CD overexpression have been shown to completely inhibit lung metastasis in murine models spontaneously metastatic breast cancer [40, 41]. Human Multiple studies have demonstrated tremendous induction of FAK expression in invasive breast cancer compared to patient-matched samples of normal breast tissue [42-46]. Impressively, all of theses studies demonstrated FAK overexpression in greater than 75% of breast cancer patients. High FAK expression, defined as high intensity immunoreactivity in greater than 90% of cells, was associated with aggressive phenotypic changes of invasive breast carcinoma, including poor nuclear grade and HER-2 receptor positivity [35, 47]. Consistent with murine data identifying FAK as the key mediator of malignant transformation of ductal cells, FAK expression appears to be an early event in human breast tumorigenesis. While FAK is overexpressed in approximately 20% of patients with premalignant atypical ductal hyperplasia, it 16

PAGE 17

is overexpressed in greater than 60% of patients with ductal carcinoma in situ [45, 48]. FAK was also overexpressed in greater than 65% of metastatic breast cancer lesions [42, 49]. VEGFR-3 Initial Characterization, Binding Partners and Signal Transduction Vascular endothelial growth factor receptor-3 (VEGFR-3, fms-like tyrosine kinase-4, flt-4) is a class III receptor tyrosine kinase originally identified independently by Galland, et. al and Aprelikova, et. al., through polymerase chain reaction (PCR) amplification of the cDNA library of human placenta and a human erythroleukemic cell line, respectively, using degenerate primers complementary to conserved receptor tyrosine kinase motifs [50-52]. The VEGFR-3 protein is divided into two domains: a 775-amino acid, N-terminal extracellular domain consisting of seven immunoglobulin-like regions containing 12 asparagine-linked glycosylation sites and a C-terminal intracellular kinase domain further divided into two regions, TK1 and TK2, by a 65amino acid sequence [51-53]. The TK1 domain is the ATP-binding site and the TK2 domain contains the tyrosine kinase catalytic site as well as the site of receptor autophosphorylation at Y1068 [53, 54]. The VEGFR-3 receptor gene mapped to the long arm of chromosome 5 and alternative splicing of the primary VEGFR-3 gene transcript forms two distinct mature messenger RNA, 5.8kb and 4.5kb, whose translation leads to the expression of two VEGFR-3 isoforms [51, 53, 55]. The long form (VEGFR-3-L, 1363 amino acids) differs from the short form (VEGFR-3-S, 1298 amino acids) by the presence of a 65 amino acid stretch at the distal end of the cytoplasmic tail, containing three potential tyrosine phosphorylation sites at positions 1333, 1337, and 1363 [55]. Using a chimeric protein construct merging the extracellular domain of colony-stimulating factor receptor with the transmembrane and intracellular domain of VEGFR-3 (CSF17

PAGE 18

VEGFR-3 chimera), Pasujola, et. al and Fournier et. al demonstrated that ligand-induced activation of VEGFR-3 led to its physical interaction with RAS-activating adapter protein Grb2 and its physical interaction and phosphorylation of the adaptor protein Shc, which is the only described substrate for VEGFR-3 tyrosine kinase activity [56, 57]. The role of the VEGFR-3-Shc interaction remains controversial. VEGFR-3 phosphorylates Shc at tandem tyrosines 239 and 240 and as well as tyrosine 313 creating to GRB2 binding sites, further linking VEGFR-3 to the activation of the RAS-MAPK pathway but mutations of the SHC substrate residues increased the transforming capacity of VEGFR-3, indicating the SHC may be a negative regulatory role of VEGFR-3 function [58]. Using a chimeric protein construct merging the extracellular domain of epidermal growth factor receptor with the transmembrane and intracellular domain of VEGFR-3 (EGFR-VEGFR-3 chimera) in human umbilical vein endothelial cells (HUVEC), Salameh, et. al. defined the functions of individual tyrosine residues with site-directed mutagenesis [54]. In agreement with Pasujola, et. al., tyrosine 1068 was found to be the key regulator of VEGFR-3 function [53]. Mutation of Y1068 completely eliminated ligand-induced receptor phosphorylation and abrogated all VEGFR-3 functions[54]. Tyrosine 1063 promoted cell survival through its interaction with Crk I/II promoting MKK4-dependent activation of c-Jun N-terminal kinase with resultant increased expression of c-Jun[54]. Consistent with previous authors, tandem tyrosines 1230 and 1231 promoted cell proliferation, migration and survival through their association with GRB2 resulting in activation of AKT and ERK [54, 56-58]. Wang et. al. have demonstrated a critical interaction between VEGFR-3 and intergrins by showing that the interaction of soluble or immobilized extracellular matrix, collagen or fibrin, with the cognate integrins containing the 1 subunit, induced the physical association of 1 and 18

PAGE 19

VEGFR-3 resulting in ligand independent transactivation of VEGFR-3 in stably-transfected 293-VEGFR-3 cells and primary dermal microvascular endothelial cells [59]. 1-mediated ECM protein activation of VEGFR-3 promoted 293-VEGFR-3 and DMEC migration, which could be increased synergistically by the addition of the ligand VEGF-D [59]. These authors further refined their model system in lymphatic endothelial cells demonstrating that the interaction of fibronectin with its cognate receptor 51 selectively enhanced ligand-induced activation of VEGFR-3, resulting in resistance to apoptosis induced by serum starvation. In agreement with other authors, this resistance to apoptosis was related to increased activation of AKT when ligand-bound 51 interacted with VEGFR-3 [60]. Using the CSF-VEGFR-3 chimera, Borg et.al demonstrated that only the long form of VEGFR-3 was capable of transforming stably transfected Rat 2 fibroblasts in the presence of ligand stimulation, resulting in anchorage-independent growth [61]. Site-directed mutagenesis identified Y1337 as the key residue responsible for the transforming capacity of VEGFR-3-L [57]. Despite the apparent lack of transforming capacity of VEGFR-3-S, both forms of VEGFR-3 were formed tumors after subcutaneous right flank implantation of stably-transfected NIH3T3 cells in mice [57]. Ligands There are two ligands for VEGFR-3, vascular endothelial growth factor-C and vascular endothelial growth factor-D [62, 63]. Both secreted ligands form covalently linked homodimers, initially consisting of precursor proteins with large aminoand carboxy terminal ends flanking a central VEGF homology domain [64]. These precursors are proteolytically processed by plasmin and the proprotein convertases furin, PC5 and PC7, increasing the binding affinity of the ligands to VEGFR-3 [64-67]. Only the mature fully processed non-covalently-bound ligand homodimers can bind to their other cognate receptor VEGFR-2, leading to the induction of 19

PAGE 20

proliferation, chemotactic migration, elongation and branching of vascular endothelial cells, linking these ligands to angiogenesis [62, 64, 67, 68]. In mice expression of VEGF-C is both necessary and sufficient for primary lymphogenesis. Overexpression of VEGF-C leads to lymphatic vessel hyperplasia. Although VEGF-D plays an important role in lymphangiogenesis in post-gestational mice, it is not necessary for primary lymphogeneis [69]. There are no known primary human diseases resulting from somatic or acquired mutations of VEGF-C or VEGF-D. VEGFR-3 is a Key Mediator of Lymphangiogenesis In vitro VEGFR-3 transduces lymphatic endothelial cell growth, survival and migration signals through the activation of ERK 1/2 and the activation AKT/protein kinase B through a non-classical pathway in that VEGFR-3 does not directly bind the p85 subunit of PI-3 kinase [61, 70]. Inhibition of VEGFR-3 using a secreted and soluble chimeric fusion of the first three immunoglobulin like loops of VEGFR-3 with the Fc portion of IgG (sVEGFR-3-Ig) prevented VEGFR-3 transduced activation of ERK 1/2 in lymphatic endothelial cells [71]. Inhibition of VEGFR-3 signaling caused regression of developing lymphatic vessels [71]. Murine Multiple murine studies have established that VEGFR-3 is the paramount regulator of lymphangiogenesis. VEGFR-3 is expressed in the embryonic cardinal vein at E8.5 but becomes restricted to the lymphatic endothelia in adult mice [72, 73]. Using a transgenic mouse model to overexpress VEGFR-3 selective VEGF-C, Veikkola et. al, demonstrated that signaling via VEGFR-3 only is sufficient for lymphangiogenesis [74]. Stimulation of VEGFR-3 produced hyperplastic, functional lymphatic vessels, the development of which could be inhibited by concomitant overexpression of s-VEGFR-3-Ig [74]. The mouse monoclonal antibody mF4-31C1 20

PAGE 21

targets the extracellular domain of murine VEGFR-3, inhibiting ligand binding and receptor phosphorylation. In an adult mouse model of skin regeneration, inhibition of VEGFR-3 activation using this antibody completely inhibited normal lymphangiogenesis in the region of regeneration and prevented exogenous VEGF-C-induced lymphatic vessel hyplerplasia [75]. Inhibition of VEGFR-3 in this model system failed to significantly suppress vascular angiogenesis in the region of skin regeneration [75]. A transgenic mouse model of tissue specific inhibition of VEGFR-3 activation demonstrated that VEGFR-3 inactivation resulted in suppression of lymphangiogenesis, apoptosis of lymphatic endothelial cells leading to regression of mature lymphatic vessels, and the development of lymphedema in the distal extremities without affecting angiogenesis or vascular endothelial function. Human Inactivating mutations of VEGFR-3 results in autosomal dominant congenital lymphedema (Milroys disease), characterized by chronic limb swelling due to lymphatic vessel dysfunction [76]. Genetic analysis of affected individuals has found that many of the mutations occur in the catalytic tyrosine kinase domain (residues 1009-1165) particularly in the consensus sequence in the central part of the catalytic loopHRDLAARNand the ATP-binding site (residues 843-943) [76, 77]. VEGFR-3 Functions in Blood Vessel Formation There are very few studies examining the role of VEGFR-3 in blood vessel formation and maturation. Persaud, et. al. demonstrated that VEGFR-3 activation mediates chemotactic migration, invasion, and tube formation of bovine aortic endothelial cells and human umbilical vein endothelial cells in an in vitro model of angiogenesis using a collagen-based three-dimensional matrix [78]. VEGFR-3 expression begins during mouse embryonic day 8.5 in developing blood vessels and is necessary for the remodeling of the primary vascular networks 21

PAGE 22

and completion of gestation [79]. Disruption of VEGFR-3 signaling in mice leads to embryonic death at day 10.5 due to cardiovascular failure [79]. VEGFR-3 and Breast Cancer In vitro The overwhelming majority of studies examining the lymphangiogenic signaling system in breast cancer have exclusively focused on the transcription, expression, and actions of the ligands VEGF-C and VEGF-D. In fact only three papers have evaluated VEGFR-3 gene transcription and/or expression in breast cancer cell lines in vitro. Among this small group of studies results are inconsistent. Using RT-PCR Akahane et. al, demonstrated VEGFR-3 mRNA transcripts in MCF7 but not in MDA-MB-231 breast cancer cells [80]. These results are contradicted by Timoshenko et, al. who failed detect VEGFR-3 mRNA transcripts in MCF7, but confirmed the absence of VEGFR-3 mRNA in MDA-MB-231 and Hs578T cells [81]. Both groups failed to correlate VEGFR-3 gene transcription with true VEGFR-3 expression. Our group demonstrated moderate to high VEGFR-3 expression in BT474 and BT20 breast cancer cells and very low receptor expression in MCF7 cells by Western blot analysis [82]. Murine Inhibition of VEGFR-3 using mF4-31C1 significantly decreased regional lymph node metastasis and total axillary lymph node tumor burden in a mouse model of spontaneously metastasizing breast cancer using orthotopic implantation of a VEGF-C overexpressing human breast cancer cell line MDA-MB-435 [83]. Reduction of regional lymph node metastasis and tumor burden occurred through blocking the paracrine effects of VEGF-C on lymphatic and vascular endothelial cell VEGFR-3 resulting in the elimination of intratumoral lymphangiogenesis, peritumoral lymphangiectasia, and the reduction of tumor-associated angiogenesis [83]. VEGFR-3 blocking antibody moderately attenuated xenograft growth but, 22

PAGE 23

VEGFR-3 expression was not evaluated in this breast cancer cell line. So it remains unclear whether the effects of the blocking antibody were due to a direct anti-proliferative action on primary tumor cells or due to the inhibition of angiogenesis required for tumor growth (Folkmann 1996). Human Gunningham et. al were the first to evaluate VEGFR-3 expression in surgically-resected specimen of invasive human breast carcinoma using an RNA protection assay of 61 snap-frozen tissues[84]. Although this study failed to demonstrate a difference in overall VEGFR-3 transcripts between neoplastic and normal tissue, it did demonstrate in a significant reduction in the long form of VEGFR-3 in breast cancer tissues. VEGFR-3 transcripts were not significantly associated with lymph node, disease-free survival or overall survival [84]. In another study of clinically resected surgical specimens, Bando, et. al detected VEGFR-3 expression in 193 patients with invasive ductal breast carcinoma by ELISA of homogenized resected specimen. VEGFR-3 expression was not significantly associated with nodal status, hormone receptor status, recurrence or nuclear grade [85] This methodology does not distinguish between VEGFR-3 derived from tumor-associated endothelial cells and VEGFR-3 derived from primary tumor cells [85]. These authors also failed to compare VEGFR-3 protein levels in patients with breast cancer to normal controls. Of the 5 studies evaluating breast cancer associated VEGFR-3 expression by immunohistochemistry, only one study detected VEGFR-3 in malignant breast cells [86-88]. In immunohistochemical studies of paraffin-embedded surgical resections from 177 unique breast cancer patients, Mylona, et. al. reported positive immunoreactivity for VEGFR-3 in the cytoplasm and the nuclei of malignant cells in 44.9% and 41.4% of the cases, respectively[88]. Detection of VEGFR-3 was not significantly correlated with stage, histologic type, nuclear 23

PAGE 24

grade, tumor size, or lymph node involvement [88] These authors do not evaluate intensity of VEGFR-3-positive immunoreactivity, the level of VEGFR-3 expression in normal tissue, or the level of receptor activation. Taken together, these in vitro, murine, and clinical show that the biological role and impact of VEGFR-3 in breast cancer remains largely unexplored. VEGFR-3 Ligands and Breast Cancer Both VEGF-C and VEGF-D are expressed in varying amount in several breast cancer cell lines. In vitro experiments have demonstrated that forced overexpression of ligand by stable transfection of expression plasmids increased breast cancer cell motility and survival under stress conditions [80, 81, 89]. The effect of ligand expression on breast cancer cell proliferation is controversial [89-91]. Murine experiments using xenotransplantation of human breast cancer cell lines have demonstrated that forced overexpression of VEGF-C or VEGF-D promotes intratumoral lymphangiogenesis, hyperplasia of peritumoral lymphatic vessels through increased lymphatic endothelial cell proliferation, and increases intralymphatic tumor emboli leading to increased lymph node metastasis [90-92]. The effect of ligand overexpression on primary xenograft growth remains controversial [90-92]. Neither VEGFR-3 nor VEGFR-2 expression are evaluated in any of the breast cancer cell lines, making it impossible to judge if ligand overexpression has any specific autocrine effects on primary xenografts [90, 92]. Although both VEGF-C and VEGF-D are also ligands for angiogenesis-associated VEGFR-2, forced ligand overexpression in breast cancer xenografts had no effect on tumor-associated blood vessels [90, 92]. The lack of effect on tumor-associated angiogenesis may be due to predominant overexpression of the unprocessed forms of the ligands, which demonstrate lower VEGFR-2 binding affinities [92]. Most of the studies evaluating ligand expression in breast cancer in humans have focused on VEGF-C. Immunohistochemical analysis of surgically-resected specimen revealed increased 24

PAGE 25

VEGF-C immunoreactivity in 40-50% of breast cancer patients when compared to patient-matched normal breast tissue [88, 93, 94] (Zhang 2008). VEGF-C overexpression was significantly associated with increased lymphatic vessel density and lymph node metastasis [93]. Multivariate Cox regression analysis revealed VEGF-C overexpression as an independent prognostic indicator of disease-free patient survival but not overall patient survival [88, 93]. VEGFR-3 and Tumor-associated Angiogenesis Using the mouse monoclonal antibody mF4-31C1, Roberts, et. al. demonstrated decreased tumor-associated angiogenesis, leading to suppression of breast cancer xenograft growth [83]. These results were corroborated by Laakonen, et.al. who demonstrated that inhibition of VEGFR-3 signaling with a similar antibody resulted in significant decreases in xenograft-associated angiogenesis with resultant increased areas of xenograft hypoxia and necrosis [95]. Both antibodies are specific to murine VEGFR-3, thus eliminating any possibility that these effects were due to actions on the primary tumor cells. VEGFR-3 expression becomes up-regulated in the endothelium of newly-formed blood vessels in human breast cancer [86, 96, 97]. FAK and VEGFR-3 Bind in Breast Cancer Cells Through phage display using the carboxy-terminus of FAK as bait, co-immunoprecipitation from whole cell lysate, far Western analysis using region-specific FAK constructs, and NMR analysis (unpublished data), Garces, et. al., recently demonstrated that VEGFR-3 physically interacts with the focal adhesion targeting domain of FAK in breast cancer cells [82]. I hypothesized that FAK and VEGFR-3 interact to transduce essential survival signals in breast cancer cells and that disruption of this interaction will decrease breast cancer cell growth and survival. 25

PAGE 26

Given the sparse amount of available data, addressing this hypothesis first required an exploration of the biological role of VEGFR-3 overexpression in breast cancer (Aim 1). Chapter 2 addresses this question using two model systems. In one model system VEGFR-3 is overexpressed in a breast cancer cell line with low endogenous expression. In the other model system VEGFR-3 expression is downregulated using VEGFR-3 specific siRNA in a cell line with high endogenous VEGFR-3 expression. Combined, these two model systems demonstrated that VEGFR-3 overexpression has a profound impact on breast cancer cells, promoting cell proliferation, motililty, invasion, survival, anchorage-independent growth and, most importantly, breast cancer cell tumorigenicity. Many of these biological effects of VEGFR-3 might be related to its binding of FAK. Chapter 3 addresses this question by demonstrating the in vitro and in vivo effects of a small molecule that disrupts that interaction between FAK and VEGFR-3 (Aims 2 and 3). Disruption of the interaction between FAK and VEGFR-2 causes dose-dependent dephosphorylation of both proteins, resulting in decreased breast cancer cell proliferation and motility, followed by the induction of apoptosis in a VEGFR-3 specific manner. In vivo, therapy with this small molecule suppresses breast cancer xenograft growth. 26

PAGE 27

CHAPTER 2 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-3 PROMOTES BREAST CANCER CELL PROLIFERATION, MOTILITY, AND SURVIVAL IN VITRO AND TUMOR FORMATION IN VIVO Abstract Vascular endothelial growth factor receptor-3 (VEGFR-3, flt-4) is a receptor tyrosine that is overexpressed in a variety of human carcinomas, but its role in carcinogenesis is not fully elucidated. Here, we characterized the effects of VEGFR-3 overexpression in human breast cancer cells using two model systems. First, we chose a cell line with low endogenous VEGFR-3 expression, MCF7, and constructed stable transfectants overexpressing VEGFR-3 called MCF7-VEGFR-3. In parallel, we used siRNA to downregulate VEGFR-3 expression in a cell line with high endogenous VEGFR-3 expression, BT474. VEGFR-3 overexpression increased cellular proliferation by 60% increase when MCF7-VEGFR-3 cells were compared to control transfectant MCF7-pcDNA3 cells. Proliferation was reduced by 42% when VEGFR-3 was downregulated in BT474 cells. VEGFR-3 overexpression promoted a three-fold increase in motility and invasion when MCF7-VEGFR-3 cells were compared to MCF7-pcDNA3 cells and motility was inhibited by downregulation of VEGFR-3 in MCF7-VEGFR-3 and BT474 cells. VEGFR-3 overexpression promoted cellular survival under stress conditions induced by staurosporine treatment. MCF7-VEGFR-3 cells formed both a greater number and larger colonies when compared to MCF7-pcDNA3 cells in a colony formation assay. Importantly, VEGFR-3 overexpression increased tumor formation, evidenced by a tumor incidence of 93% in MCF7-VEGFR-3 xenografts compared to no tumors formed by MCF7-pcDNA3 xenografts in the absence of hormonal stimulation. With estrogen stimulation, MCF7-VEGFR-3 xenografts were ten times larger in volume than MCF7-pcDNA3 xenografts. 27

PAGE 28

To our knowledge, we are the first to demonstrate that VEGFR-3 overexpression significantly promotes breast cancer cell proliferation, motility, survival, anchorage-independent growth and tumorigenicity in the absence of ligand expression. Introduction The vascular endothelial growth factor receptors (VEGFRs) are a subfamily of receptor tyrosine kinases that play key roles in angiogenesis and lymphangiogenesis. VEGFR-1 (flt-1) and VEGFR-2 (flk-1/kdr) are primarily located on vascular endothelial cells and when activated by their cognate ligands play a major role in tumor angiogenesis [1]. VEGFR-3 (flt-4) is primarily located on lymphatic endothelial cells and, along with its ligands VEGF-C and VEGF-D, represents the most extensively studied lymphangiogenic signaling system in cancer [2]. Activation of the VEGF-C/VEGF-D/VEGFR-3 axis through ligand overexpression induces intratumoral lymphangiogenesis, peritumoral lymphatic hyperplasia, and/or increased lymph node metastasis in animal models for carcinoma of the breast [4, 5], stomach[6], rectum[7], lung[8, 9] and skin[10]. These model systems demonstrate the extensive paracrine effects of tumor ligand secretion on lymphatic endothelia, including increased lymphatic endothelial cell size, proliferation and vessel permeability leading to lymphatic metastasis of primary tumor xenografts[12, 13]. In humans, up-regulation of the VEGF-C/VEGF-D/VEGFR-3 axis through ligand overexpression promotes tumor invasion and increases lymph node metastases in gastric [14] and colorectal adenocarcinoma [15] and is an independent indicator of poor prognosis in endometrial[16], ovarian[17], and esophageal squamous cell carcinoma[18]. Despite this abundance of correlative animal xenograft and clinical data regarding ligand overexpression, very little is known about the actual biological effects of VEGFR-3 overexpression on primary tumors. 28

PAGE 29

VEGFR-3 is overexpressed in the primary tumor cells of a subset of patients with gastric adenocarcinoma[19], colorectal adenocarcinoma[15, 20], endometrial carcinoma [16], and ovarian carcinoma [17]. The biological effects of VEGFR-3 overexpression on cancer progression vary with tumor type. Shida et. al., reported that VEGFR-3 overexpression did not effect tumor invasion or lymphatic metastasis in gastric adenocarcinoma [19]. Yokoyama et. al., have shown VEGFR-3 overexpression to be a significant promoter of lymphatic metastasis in ovarian and endometrial carcinoma [16, 17]. However, these results are complicated by concomitant tumor overexpression of the ligand VEGF-D. The impact of VEGFR-3 overexpression in colorectal adenocarcinoma remains controversial [21, 22]. We have previously demonstrated VEGFR-3 overexpression in the primary tumor cells of patients with invasive ductal breast adenocarcinoma [23]. In this study we focused on defining the biological effects of VEGFR-3 overexpression in human breast cancer cells. To our knowledge, this is the first report to address the significance of VEGFR-3 overexpression in the absence of ligand overexpression. We have shown that VEGFR-3 overexpression significantly increases the proliferation, motility, viability and tumorigenicity of human breast cancer cells. Materials and Methods Cell lines MCF7 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The BT474 cells are a subclone of the original cell line that does not express the receptor tyrosine kinase Her-2/neu. BT474 were maintained in RPMI-1640 with 10% fetal bovine serum and insulin 250 ug/ml. MCF7 cells were maintained with Modified minimum Eagles media with 10% fetal bovine serum, 1X non-essential amino acids (Cellgro, Herndon, VA, USA), 1 mM sodium pyruvate, and 500g/ml insulin. 29

PAGE 30

Antibodies and Reagents Santa Cruz Biotechnology, Inc (Santa Cruz ,CA, USA): VEGFR-3 C-20 (sc-321) Calbiochem (San Diego, CA, USA): p-VEGFR-3 (pc460) Cell Signaling Technology (Danvers, MA, USA): Pro-caspase-8 (#9746), erk 1,2 (#9102), p-erk (#4377S), akt (#9272), p-akt (9271S), PARP (#9542) W estern Blot Appropriately treated or non-treated cells were allowed to grow until they were 80-85% confluent or until treatment was completed. Cells were twice washed with ice-cold phosphate-buffered saline (PBS), then incubated on ice with 1% NP-40 lysis buffer with inhibitors mM NaCl, 20 mM Tris-Base, pH 7.4, 5 mM EDTA 1% Nonidet P-40, 50mM NaF, 10mM NaVO 4 100 L phosphatase inhibitor cocktail I and II, according to the manufacturers instructions. (Calbiochem, San Diego, CA, USA), 200 L Complete Protease Inhibitor, according to the manufacturers instructions. (Roche Diagnostics, Mannheim, Germany)for 30 minutes. Plates were scraped and lysate was centrifuged at 14,000 rpm for 30 minutes at 4 C. Protein concentration of cell lysate was measured in duplicate using the colorimetric BCA Protein Assay Kit (Pierce, Rockford, IL, USA) with bovine serum albumin as a standard. The appropriate amount of Laemmli loading buffer was mixed with protein lysate and boiled for 5 minutes at 95 C. Fifty micrograms of total protein were loaded and resolved by SDS-PAGE using 7.5%, 10% or 4-20% Tris-Hcl gel (Biorad, Hercules, CA, USA). Resolved bands were transferred onto PVDF membrane for two hours at 80 mV at room temperature. PVDF membrane was then blocked with 5% BSA in tris-buffered saline tween (TBST, 25mM Tris-Hcl, 125 mM NaCl, 0.1% tween) for one hour. The appropriate primary antibody diluted in 5% BSA-TBST (1:1000) was added for two hours at room temperature. Membrane was then washed three times for five minutes each with TBST. The appropriate secondary antibody conjugated with 30

PAGE 31

horseradish peroxidase, diluted in 5%BSA-TBST was added for one hour at room temperature. Membranes were washed three times with TBST, then developed using Western Lightning Chemilumnescence Reagent Plus (Perkin Elmer, Boston, MA, USA). Membrane was stripped using Restore Western Blot Stripping Buffer (Pierce, Rockford, IL, USA) for 15 minutes at 37C, then washed three times in TBST for five minutes each wash and reused. Transfection Stable transfection of MCF7 cells was performed using SuperFect transfection reagent (Qiagen, Valencia, CA, USA), according to the manufacturers protocol. 5 X 10 5 cells were plated on a 60-mm plate and allowed to attached overnight. 5 g of each plasmid expression vector was added to serum-free Eagles minimum essential media (DMEM, Mediatech, Inc., Herndon, VA, USA) to make the total volume 150 l. 60 l of SuperFect tranfection reagent was added to the plasmid solution and mixed by pipetting. This reaction mixture was incubated for 10 minutes. During incubation MCF7 were washed with 4 ml of 1X phosphate-buffered saline (PBS, Mediatech, Inc,). 1 ml of MCF7 growth media was added to the reaction mixture. Complete mixture was added to washed MCF7 cells and incubated at normal growth condition (37C, 5% CO2) for 2.5 hours. Transfected cells were washed with 7 ml of 1X PBS. 10 ml of MCF7 growth media was added to each plate. Cells grown under normal growth conditions for 24 hours. Cells were then washed with 10 ml of 1X PBS and 10 ml of MCF7 growth media was added supplemented with G418 (MP Biomedicals, Inc., Solon, Ohio, USA) at a concentration of 500 g/mL. (kindly provided by Hiroyuki Suzuki, Ph.D. University of Tsukuba, Tsukuba, Japan) BrdU Incorporation Assay BrdU incorporation was performed using BrdU Cell Proliferation Assay, HTS (Calbiochem, San Diego, CA, USA), according to the manufacturers protocol. 2.5 X 10 3 cells 31

PAGE 32

were plated into a 96-well plate and allowed to attach overnight. 100 l of fresh growth media or growth media with treatment was added to each well followed by 20 l of BrdU labeling solution. Cells were incubated for 24 hours. Labeling solution was removed and 200 l of the fixative solution was added for 30 minutes at room temperature. 100 l of anti-BrdU antibody solution was added to each well and incubated for 1 hour at room temperature. Cells were washed three times with Wash Buffer. 100 l of peroxidase Goat Anti-Mouse IgG HRP conjugate solution was added and incubated for 30 minutes at room temperature. Cells were washed three times with Wash Buffer. Wells were flooded with dH 2 O. 100 l of Fluorogenic Substrate Working Solution is added to each well and incubated at room temperature for 30 minutes. 100 l of Stop Solution was added to each well. Plates were read by fluorometer at 320 nm / 460nm. Immunocytochemistry 3 X 10 4 cells were plated in 24-well plates with a cover slip placed at the bottom of the well. Cells on the coverslip were washed three times with PBS. Cells were fixed by adding 3.7% formaldehyde in PBS for 15 minutes, washed three times with PBS, permeabilized with 0.1% Triton-X100 in PBS for 3 minutes, washed three times with PBS, blocked with 10% normal goat serum (NGS) in 2%BSA-PBS for 1 hr, washed three times with PBS, incubated with primary antibody at a concentration of 5 g/mL in 2%BSA-PBS for 1 hour, washed three times with PBS., ncubated with the appropriate secondary antibody-fluorescent molecule conjugate, Alexa-488 (Invitrogen, Carlsbad, CA, USA), diluted 1:100 in 10% 2%BSA-PBS for 45 minutes in the dark. Cells were washed three times with PBS. Cells on cover slip were mounted to slides using Vectashield Hard Set mounting medium (Vector Laboratories, Burlingame, CA, USA).with DAPI. 32

PAGE 33

MTT (Cell Viability) Assay. 5.0 X 10 3 (100 L) were plated in 96-well plates and were allowed to attach overnight. One hundred microliters of fresh media with or without staurosporine was added to each well. Cells were treated for designated amount of time. Each well was incubated with twenty microliters of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) for 1-2 hours. Colorimetric changes were read at wavelength of 490 nm using Benchmark microplate reader (Biorad, Hercules, CA, USA). Motility and Invasiveness Assay using Matrigel Modified Boyden Chamber Protocol was performed using BD bioCoat Matrigel Invasion Chamber (BD Biosciences, Bedford, MA, USA). Cells were grown overnight in serum-reduced growth media with 1%FBS. Matrigel chambers were rehydrated for two hours using 500 l of serum-free media. After rehydration, 750 l normal growth media containing 10% FBS was added to the bottom of the well. 5.0 X 10 4 cells in serum-reduced growth media (500 ul) were added to the matrigel chamber. Chambers were incubated overnight in a cell culture incubator, at 37C, 5% CO 2 atmosphere. Non-invading cells were removed with a cotton swab. Diff-Quik solution was added to a 24-well plate. Matrigel chambers were sequentially transferred through a fixative, then two staining solutions, followed by two beakers of double distilled water and were allowed to air dry. Membranes were mounted onto slides using immersion oil and covered with a cover slip. Cells were counted using light microscopy at 40X magnification. RT-PCR Protocol was performed using Cloned AMV First-Strand cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA). 10 M VEGFR-3 primer (proprietary sequence of R&D Systems, RDP-108-025) was combined with 5 g of RNA and 10 M dNTP mix. 8 l of master mix (cDNA synthesis buffer, 0.1 M DTT, RNaseOUT, DEPD-treated water, Cloned AMV 33

PAGE 34

reverse transcriptase) was added to the PCR tube on ice. Reaction was incubated at 50C for 1 hour. Reaction was terminated by incubating at 85C for 5 minutes. CDNA, primers, dNTP, Herculase are mixed. PCR was performed in the following manner: 10 cycles of 95C for 30 seconds, 55C for 30 seconds, 72C for 70 seconds; 30 cycles of 95C for 30 seconds, 55C for 30 seconds, 72C for 80 seconds. Colony Formation Assay 1.5 mL 2X MCF7 growth media was combined with 1.5 mL of 1% bact-agar solution, plated onto 60 mm petri dish and allowed to solidify, creating the bottom layer of 0.5% bact-agar-1x MCF7 growth media. 1 X 10 4 cells in 1.5 mL 2X MCF7 growth media was combined with 1.5 mL of 0.7% agarose solution and plated on top of the bottom layer. After 14 days, plates were stained with 3 ml of 0.005% crystal violet for 3 hours. Plates were divided into quadrants. Colonies were counted in the two quadrants with the highest colony densities. Animal Model Subconfluent MCF7-pcDNA3 and MCF7-VEGFR-3 were collected by centrifugation after treatment with EDTA-trypsin. Cells were washed twice in 1X PBS, then diluted in sterile 1X PBS to a concentration of 1 X 10 6 cells per 100 l. In accordance with the University of Florida IACUC approved protocol E557, 2 X 10 6 cells (200 l) were subcutaneously injected into the right flank of the 6-week old balbc nu/nu mice. Tumor volume was measured thrice weekly using the formula length X width 2 X 0.5. After three weeks animals were sacrificed. Animals were housed in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture. 34

PAGE 35

Statistical Analysis Data presented are the means and 95% confidence intervals of the three or more experiments. For in vitro and in vivo experiments comparison between groups were made using a two-tailed two-sample Students t test. Differences for which P value was less than 0.05 were considered statistically significant. Results Establishment of Model Systems to Examine the Effects of VEGFR-3-overexpression in Breast Cancer. To characterize the biological effects of VEGFR-3 overexpression in human breast cancer cells, we established two parallel model systems based on endogenous VEGFR-3 expression. MCF7 breast cancer cells express very low levels of endogenous VEGFR-3. As our first model, we stably transfected MCF7 cells with a VEGFR-3 expression plasmid, creating the modified cell line called MCF7-VEGFR-3. As a control for this model, we stably transfected the same MCF7 cells with an empty expression vector, pcDNA3.1B called MCF7-pcDNA3. These cell lines allowed us to examine the biological effects of VEGFR-3 overexpression in this breast cancer cell line. In parallel, we chose a breast cancer cell line with high endogenous expression of VEGFR-3, BT474, and established a model of receptor downregulation using VEGFR-3-specific siRNA. We demonstrated VEGFR-3 mRNA transcripts in BT474 and MCF7-VEGFR-3 cell lines by RT-PCR with VEGFR-3-specific primers (Figure 2-1A). Parental MCF7 cells and control MCF7-pcDNA3 cells demonstrated very low detectable amounts of VEGFR-3 transcripts (Figure 2-1A). Consistent with transcript level, Western blot analysis revealed that both BT474 and MCF7-VEGFR-3 abundantly expressed VEGFR-3, which was highly phosphorylated (Figure 2-1B). In contrast, the expression of VEGFR-3 was detected in very low amounts in 35

PAGE 36

parental MCF7 and MCF7-pcDNA3 cells (Figure 2-1B). We demonstrated subcellular localization of VEGFR-3 to the perinuclear and cytoplasmic regions of MCF7-VEGFR-3 and BT474 cells using immunofluorescence microscopy with a VEGFR-3 specific primary antibody (Figure 2-1C). Confirming our expression data, VEGFR-3 was not detected in MCF7-pcDNA3 cells by this method (Figure 2-1C). These results established our model of VEGFR-3 overexpression in MCF7 cells. In our second model system, VEGFR-3 downregulation in BT474 using receptor specific siRNA was shown by Western blot analysis that demonstrated dose-dependent reduction of VEGFR-3 expression in BT474 cells (Figure 2-1D). Since VEGFR-3 overexpression is frequently associated with overproduction of its cognate ligands [17]we analyzed production of the ligands VEGF-C and VEGF-D by ELISA of conditioned media from each cell line in this study. The amount of each ligand in the conditioned media was below the minimum detectable dose (13.3 pg/mL) for each cell line (data not shown). This data regarding the absence of ligand expression correlates well with data shown by Akahane, et. al, who demonstrated VEGF-D transcripts but the absence of expression or secretion of VEGF-D in MCF7 cells and Mattila, et.al. who demonstrated lack of VEGF-C mRNA transcripts or protein expression in this cell line [23]. We concluded that neither overexpression of VEGFR-3 in MCF7 cells nor downregulation of VEGFR-3 in BT474 cells led to the activation of an autocrine loop through increased ligand expression. Thus, we established two parallel model systems: increased expression of VEGFR-3 in MCF7 cells using stable plasmid transfection and downregulation of VEGFR-3 in BT474 cells using siRNA. These models allowed us to examine the biological effects of VEGFR-3 overexpression in these human breast cancer cell lines in the absence of ligand secretion. 36

PAGE 37

VEGFR-3 Increases Breast Cancer Cell Viability and Proliferation. Our first experiment evaluated the impact of VEGFR-3 overexpression on the baseline characteristics of MCF7 and BT474 cells. MCF7-VEGFR-3 cells demonstrated a 36% increase in cellular viability compared to control MCF7-pcDNA3 cells after 24 hours of growth (Figure 2-2A). Similarly, downregulation of endogenous expression of VEGFR-3 with VEGFR-3-specific siRNA in BT474 cells resulted in a 42% reduction in cellular viability when compared with control siRNA-treated BT474 cells (Figure 2-2B). Since MTT assays are broad indicators of cellular activity, we specifically confirmed the impact of VEGFR-3 overexpression on cellular proliferation using a BrdU incorporation assay. In our VEGFR-3 overexpression model, there was a 60% increase in DNA synthesis after 24 hours when MCF7-VEGFR-3 cells were compared to MCF7-pcDNA3 cells (Figure 2C). In our model of receptor downregulation, treatment of BT474 with VEGFR-3 specific siRNA resulted in a 32% reduction in DNA synthesis when compared to control siRNA treated cells after 24 hours of growth (Figure 2-2D). Taken together, these experiments demonstrate that VEGFR-3 is involved in the regulation of cell viability and its overexpression significantly increases proliferation in human breast cancer cells. Overexpression of VEGFR-3 Increases Breast Cancer Cell Motility and Invasion. Next, we continued to characterize the biological effects of VEGFR-3 overexpression by examining its effects on cellular motility. In our VEGFR-3 overexpression model, increased receptor expression in MCF7-VEGFR-3 cells resulted in a 3.5 fold increase in motility as compared to MCF7-pcDNA3 cells (Figure 2-3A). Furthermore, we downregulated VEGFR-3 in MCF7-VEGFR-3 cells using siRNA, resulting in a three-fold reduction in motility when compared to untreated and control siRNA-treated MCF7-VEGFR-3 cells (Figure 2-3B). Finally we used our model of endogenous VEGFR-3 downregulation in BT474 cells. Treatment of 37

PAGE 38

BT474 cells with VEGFR-3 specific siRNA resulted in a 2.5-fold decrease in motility when compared to untreated and control siRNA-treated BT474 cells (Figure 2-3C). Treatment of BT474 with control siRNA resulted in a non-significant reduction in BT474 cell motility. Overexpression of VEGFR-3 in MCF7-VEGFR-3 also increased the invasive potential of MCF7 cells, resulting in a four-fold increase in the percentage of MCF7-VEGFR-3 cells capable of invading through a matrigel matrix when compared to MCF7-pcDNA3 cells (Figure 2-3D). These results demonstrated that VEGFR-3 overexpression plays a significant role in promoting breast cancer cell motility and invasion. VEGFR-3 Promotes Survival in Breast Cancer Cells Under Stress Conditions. Overexpression of many receptor tyrosine kinases often leads to resistance to apoptosis and promotes cell survival in stress conditions [25]. To evaluate the effect of VEGFR-3 overexpression on cellular survival under stress conditions, we treated MCF7-VEGFR-3 and MCF7-pcDNA3 cells with the broad-range kinase inhibitor, staurosporine. MTT assays revealed that after staurosporine treatment approximately 47% more MCF7-VEGFR-3 cells were viable when compared to MCF7-pcDNA3 cells (Figure 2-4A). As expected, treatment with staurosporine dephosphorylated VEGFR-3 in MCF7-VEGFR-3 cells, demonstrated by Western blot anaylsis (Figure 4B). This analysis also revealed that decrease in viability of our control cells related to the induction of apoptosis evidenced by cleavage (activation) of pro-caspase 8 and PARP. Moreover, resistance to apoptosis was indeed associated with VEGFR-3 overexpression, because caspase 8 was activated in MCF7-pcDNA3 cells but not in MCF7-VEGFR-3 cells (Figure 2-4B). Additionaly, biochemical analysis revealed that the basal level of PARP expression in MCF7-VEGFR-3 was approximately 3 times greater than the basal level of PARP in MCF7-pcDNA3. We estimated the ratio of cleaved to uncleaved PARP in staurosporine treated cell 38

PAGE 39

lines using densitometry and found that MCF7-VEGFR-3 cells treated with STS have less cleaved PARP (1:0.59 ratio) than the MCF7-pcDNA3 cells (ratio of 1:1.2). Together with the pro-caspase-8 activation data, this difference in PARP cleavage indicates that overexpression of VEGFR-3 promotes breast cancer cell survival under stress conditions. This resistance to apoptosis might be due to the association of VEGFR-3 overexpression with increased phosphorlyation (activation) of AKT and ERK (Figure 2-4B.) VEGFR-3 Promotes Anchorage-independent Growth. Thus far, our data have shown that VEGFR-3 overexpression in human breast cancer cells increased proliferation, motility and survival in vitro. Next, we wanted to evaluate the effect of VEGFR-3 overexpression on breast cancer cell tumorigenicity. We used a colony-formation assay to test the effect of VEGFR-3 overexpression on anchorage-independent growth. After 14 days of incubation in soft agar, significantly more colonies were formed by MCF7-VEGFR-3 cells when compared to MCF7-pcDNA3 cells (15642 versus. 2213; p<0.001, Figure 2-5B). Not only did VEGFR-3 overexpression lead to increased colony formation, but also the size of the largest colonies formed by MCF7-VEGFR-3 was significantly larger than those formed by MCF7-pcDNA3 with a mean diameter 5.5 times greater (Figure 2-5A). These in vitro data indicated that VEGFR-3 overexpression might impact tumorigenicity of MCF7 cells in vivo. VEGFR-3 Promotes Growth of Breast Tumors in an Animal Model. Our final assessment of the biological effect of VEGFR-3 overexpression in human breast cancer cells focused on the tumorigenicity of MCF7 cells in a xenotransplant nude mouse model. Previous data have shown that MCF7 cells are non-tumorigenic with both subcutaneous and orthotopic modes of implantation without hormonal stimulation [24, 26]. In order to investigate the in vivo biological effects of VEGFR-3 overexpression on tumor formation, we 39

PAGE 40

subcutaneously implanted 2.0 X 10 6 MCF7-pcDNA3 and MCF7-VEGFR-3 into the right flank of nude mice in the absence of hormonal stimulation. Twenty-one days after implantation only MCF7-VEGFR-3 implants formed tumors (Figure 2-6B.) These tumors had a mean tumor volume of 2153395 mm 3 To drive tumor formation of MCF7-pcDNA3 cells xenografts, we implanted nude mice with estrogen pellets one week prior to breast cancer cell implantation, which increased the tumor incidence of MCF7-pcDNA3 xenografts to 100%. Consistent with our tumorigenicity data in the absence of hormonal stimulation, MCF7-VEGFR-3 xenografts grew at a much faster rate compared to the MCF7-pcDNA3 xenografts (Figure 2-6A). Even in the presence of hormonal stimulation MCF7-VEGFR-3 xenografts were ten times larger in volume than the MCF7-pcDNA3 xenografts three weeks after implantation; MCF7-pcDNA3 vs. MCF7-VEGFR-3, 239 mm 3 vs 2589 mm 3 respectively (p<0.01, Figure 2-6B). Representative tumors for each group eighteen days after implantation are shown in Figure 2-6C. Tumor lysate from selected harvested xenografts were evaluated by Western blot analysis demonstrating overexpression of highly phosphorylated VEGFR-3 in MCF7-VEGFR-3 xenografts compared to the absence of VEGFR-3 expression in MCF7-pcDNA3 xenografts (Figure 2-6C). From these in vivo experiments we concluded that VEGFR-3 overexpression significantly impacted the tumorigenicity of breast cancer cells, leading to the increased formation of rapidly growing xenografts. Immunohistochemical analysis of these breast cell xenografts revealed significantly increased peritumoral lymphangiogenesis associated with MCF7-VEGFR-3 xenografts as compared to MCF7-pcDNA3 xenogratfs when samples were stained with LYVE-1, an antibody that specifically detects a lymphatic endothelial cells (Figure 2-6D;p<0.001). This increased 40

PAGE 41

peritumoral lymphangiogenesis might impact the metastatic potential of the MCF7-VEGFR-3 xenograft. Discussion VEGFR-3 is overexpressed in a variety of human tumors, but its role in carcinogenesis has not been fully elucidated. In this study, we demonstrated that VEGFR-3 overexpression had a tremendous impact on several important biological characteristics of human breast cancer cells. VEGFR-3 overexpression promoted proliferation, motility, survival, and, most importantly, tumorigenicity of MCF7 and BT474 cells. The salience of these results is increased by the fact that neither ligand VEGF-C nor VEGF-D was concomitantly overexpressed in these cell lines. To our knowledge, this is the first study to focus solely on VEGFR-3 overexpression and its biological impact on breast cancer. Since the landmark papers by Skobe, et. al demonstrating that overexpression of the ligand VEGF-C increased growth and lymphatic metastasis of breast cancer xenografts, the majority of the work on the VEGF-C/VEGF-D/VEGFR-3 axis has focused on these ligands. Many of the subsequent studies do not even address receptor expression or its impact on tumor biology. Our results show that overexpression solely of VEGFR-3 is at least as important as ligand overexpression, in that either phenomenon promoted characteristics associated with cancer progression, including increased proliferation, resistance to apoptosis and promotion of tumor growth. For instance, our data show that overexpression of highly phosphorylated VEGFR-3 in MCF7 cells resulted in a 60% increase in cellular proliferation at 24 hours. Akahane, et. al. have reported a similar effect when MCF7 cells were stably transfected to overexpress the ligand VEGF-D[24]. These authors also demonstrated that VEGF-D overexpression in MCF7 cells led to resistance to apoptosis induced by staurosporine, cycloheximide and oxygen deprivation [28]. 41

PAGE 42

Our data showed that overexpression solely of VEGFR-3 sufficiently promoted resistance to apoptosis that might be related to the association of receptor overexpression with the upregulation of three known cellular survival factors: PARP, AKT, ERK1/2 (Figure 2-4B). There was a large difference in basal PARP expression between our control stable clone MCF7-pcDNA3 and our VEGFR-3 overexpressing stable clone MCF7-VEGFR-3. The importance of PARP as a survival factor has been recently demonstrated. Mason et al., have shown that inhibition of PARP sensitizes breast cancer cells to chemotherapeutic agents[29]. Calabrese et al., demonstrated sensitization to radioand chemo-therapy through PARP inhibition in colorectal cancer cells[30]. Although the total amount of AKT was lower in our receptor overexpressing MCF7-VEGFR-3 cells, in these cells phosphorylation (activation) of AKT was higher compared to MCF7-pcDNA3 cells. AKT is a well-known promoter of resistance to apoptosis through its ability to phosphorylate caspase-9, preventing initiation of cell death, to phosporylate MDM2, increasing inhibition of p53 and to phosphorylate BAD, preventing mitochondrial cell death. In our study, tumor formation from MCF7-pcDNA3 xenografts varied with hormonal stimulation. Without estrogen stimulation MCF7-pcDNA3 xenografts did not grow. This is consistent with data published by Akahane et al. [24, 26]. In contrast, receptor overexpressing MCF7-VEGFR-3 xenografts formed fast-growing tumors irrespective of hormonal stimulation. The fact that VEGFR-3 overexpression promotes estrogen-independent tumor growth may have important clinical implications. In summary, VEGFR-3 overexpression has a profound impact on breast cancer cell carcinogenesis, promoting characteristics important for cell survival and tumor progression. Most importantly, VEGFR-3 overexpression increased tumorigenicity and tumor growth of 42

PAGE 43

breast cancer cells in the absence of ligand expression or hormonal stimulation. These data indicate that VEGFR-3 might be a useful therapeutic target in the treatment of breast cancer. 43

PAGE 44

BT474MCF7MCF7-VEGFR-3150 kDa150 kDaVEGFR-3P-VEGFR-3MCF7-pcDNA3B.A.MCF7-pcDNA3Vector controlMCF7-VEGFR-3MCF7 GAPDH VEGFR-3BT474 GAPDHVEGFR-3UntreatedsiControlsiVEGFR-3 10 nmD. C. GAPDH MCF7-pcDNA3MCF7-VEGFR-3 BT474siVEGFR-3 1 nm Figure 2-1. Establishment of the Model Systems. MCF7 breast cancer cells were stably transfected with expression plasmid for VEGFR-3 or an empty vector (pcDNA3) and selected for antibiotic resistance. A.) VEGFR-3 transcription. Reverse Transcription followed by polymerase chain reaction (RT-PCR) was performed with VEGFR-3-specific primers to demonstrate transcription of the VEGFR-3 gene in stably transfected MCF7 cells. BT474 cells also demonstrate VEGFR-3 gene transcription. B.) VEGFR-3 Expression in Breast Cancer Cell Lines. Western blot analysis demonstrating expression of the VEGFR-3 in stably transfected MCF7 cells. Both BT474 and MCF7-VEGFR-3 cells expresses highly phosphorylated VEGFR-3. Neither the parental cell line nor MCF7-pcDNA3 demonstrate VEGFR-3 transcription or expression. C.) Subcellular Localization of VEGFR-3. Immunofluorescent cytochemistry was performed and demonstrated perinuclear, cytoplasmic, and membrane subcellular localization of VEGFR-3 in MCF7-VEGFR-3 and BT474 cells. VEGFR-3 detected with Santa Cruz sc-321 followed by Alexa 488-conjugated rabbit secondary antibody. Nuclei stained with DAPI. D.) VEGFR-3 knockdown with siRNA. Western blot analysis demonstrating a dose-dependent downregulation of VEGFR-3 after treatment with si-RNA targeted to VEGFR-3 for 48 hours. 44

PAGE 45

A.B. MCF700.20.40.60.811.21.41.61.8MCF7-pcDNA3MCF7-VEGFR-3Cell Viability (Normalized Ratio) BT47400.20.40.60.811.2untreatedControl siRNAVEGFR-3 siRNACell Viability (Normalized Ratio) ****C.D. MCF7050010001500200025003000350040004500MCF7-pcDNA3MCF7-VEGFR-3Relative Fluorescence Units ** BT4740100020003000400050006000700080009000untreatedControl siRNAVEGFR-3 siRNAR e l at iv e Fl uo r es c e n ce U ni ts Figure 2-2. VEGFR-3 increases viability and proliferation of breast cancer cells. A.) 5 X 10 3 cells in 96-well plate and grown for 24 hours. MTT assay demonstrating a 36% increase in cellular viability of MCF7-VEGFR-3 when compared to MCF7-pcDNA3 (**, p<0.001). B.) BT474 cells treated with VEGFR-3 specific siRNA 10 nM demonstrate a 42% decrease in cellular viabilty when compared to Control siRNA-treated BT474 (**, p<0.001). C.) 2.5 X 10 3 cells plated overnight in 96-well plate. BrdU incorporation measured at 24 hours using fluorogenic enzyme immunoassay. MCF7-VEGFR-3 demonstrates a 60% increase in proliferation when compared to MCF7-pcDNA3 (**, p<0.001). D.) BT474 cells treated with VEGFR-3 specific siRNA (10 nM) demonstrate a 32% decrease in proliferation when compared to Control siRNA treated BT474 (*, p=0.0019). 45

PAGE 46

A.B. MCF700.511.522.533.544.55MCF7-pcDNA3MCF7-VEGFR-3Motility (Fold s MCF7-VEGFR-300.20.40.60.811.2untreatedControl siRNA VEGFR-3 siRNAMotility (Fold s ** C. BT47400.20.40.60.811.2Untreated Control siRNAVEGFR-3 siRNA Motility (Fold s MCF7051015202530354045MCF7-pcDNA3MCF7-VEGFR-3Percent Invasio n ** D. Figure 2-3. VEGFR-3 increases motility of breast cancer cells. 5 X 10 4 cells plated on membrane with 8-micron pores in a modified Boyden chamber. Number of cells able to traverse the membrane were measured by cell count after serial staining with DiffQuik solution. A.) MCF7-VEGFR-3 demonstrates a three-fold increase in motility when compared to MCF7-pcDNA3(*, p<0.05). B.) MCF7-VEGFR-3 cells treated with VEGFR-3 specific siRNA 10 nM demonstrate a 4-fold decrease in motility when compared to Control siRNA treated MCF7-VEGFR-3 (**, p<0.001). C.) BT474 cells treated with VEGFR-3-specific siRNA 10nM demonstrate a 2.5-fold decrease in motility when compared to Control siRNA treated BT474 (*, p<0.05). 46

PAGE 47

A. 00.20.40.60.811.2untreatedSTS 100 nMuntreatedSTS 100 nM MCF7-pcDNA3MCF7-VEGFR-3 Cell Viability (Normalized Rat ** B. akt GAPDH p-erkPro-caspase-8 p-akt p-VEGFR-3 STS-+ -+MCF7-pcDNA3 MCF7-VEGFR-3erkSTS-+ -+PARPCleaved PARP1:0.59MCF7-VEGFR-31:1.2MCF7-pcDNA3PARP:Cleaved PARP Ratio (STS Treated)Cell Type 1:0.59MCF7-VEGFR-31:1.2MCF7-pcDNA3PARP:Cleaved PARP Ratio (STS Treated)Cell Type MCF7-pcDNA3 MCF7-VEGFR-3 Figure 2-4. VEGFR-3 promotes resistance to apoptosis. A.) 5 X 10 3 cells were plated in 96 well plate, attached overnight and treated with staurosporine 100 nM for 24 hours. MCF7-VEGFR-3 cells demonstrates a 47% increase in cell viability when compared to MCF7-pcDNA3 cells after treatment with a broad-range kinase inhibitor, staurosporine measured by MTT assay (**, p<0.001). B.) Biochemical analysis demonstrates resistance to apoptosis in MCF7 cells overexpressing VEGFR-3 evidenced by Western blot analysis, which demonstrates twice as much cleaved PARP in control MCF7-pcDNA3 cells (Lanes 1 and 2) as compared to MCF7-VEGFR-3 cells (lanes 3 and 4) after treatment. PARP to cleaved PARP ratio was calculated after densitometry. Resistance to apoptosis is also indicated by lack of cleavage of pro-caspase-8 in MCF7-VEGFR-3 cells. In MCF7-VEGFR-3 cells, VEGFR-3 overexpression is associated with increased phosphorylation (activation) of AKT and ERK 1/2. 47

PAGE 48

MCF7-pcDNA3 MCF7-VEGFR-3 A.31.1**15641.9**25131MCF7-VEGFR-35.5.9722.7022MCF7-pcDNA3Mean DiameterTotal ColoniesLarge ColoniesSmall ColoniesCell Line 31.1**15641.9**25131MCF7-VEGFR-35.5.9722.7022MCF7-pcDNA3Mean DiameterTotal ColoniesLarge ColoniesSmall ColoniesCell Line B. Figure 2-5. VEGFR-3 promotes anchorage-independent growth of breast cancer cells. 1 X 10 4 cells suspended in 0.35% soft agar gel. Colonies counted in quadrants with highest density 14 days after suspension. A.) Colonies formed by MCF7-VEGFR-3 were significantly larger than colonies formed by MCF7-pcDNA3 (31.1 m vs. 5.5.97 m, ** p<0.001). Phase micrographs are representative examples of largest colonies for each cell type. B.) Colonies counted in 2 quadrants with highest density 14 days after suspension. MCF7-VEGFR-3 formed a significantly greater number of colonies when compared to MCF7-pcDNA3 (15642 vs. 2213, ** p<0.001). 48

PAGE 49

Yes2589429***5/5 (100%)MCF7-VEGFR-3Yes239795/5 (100%)MCF7-pcDNA3No2153514/15 (93%)MCF7-VEGFR-3NoN/A0/11 (0%)MCF7-pcDNA3Estrogen PelletMedian Tumor Volume (mm3)Tumor IncidenceCell Line Yes2589429***5/5 (100%)MCF7-VEGFR-3Yes239795/5 (100%)MCF7-pcDNA3No2153514/15 (93%)MCF7-VEGFR-3NoN/A0/11 (0%)MCF7-pcDNA3Estrogen PelletMedian Tumor Volume (mm3)Tumor IncidenceCell Line A.B. MCF7-pcDNA3 vs MCF7-VEGFR-3 with hormonal stimulation0500100015002000250030003500Day 2Day 6Day 8Day 11Day 13Day 15Day 18Tumor Volume (mm3) MCF7-pcDNA3 MCF7-VEGFR-3 **** C.D.MCF7-pcDNA3MCF7-VEGFR-3 Figure 2-6. VEGFR-3 increases tumor formation and tumor growth in xenotransplant mouse model. A.) Graph demonstrating tumor volume over time for MCF7-pcDNA3 and MCF7-VEGFR-3 xenografts with hormonal stimulation (*, p<0.05). 5.0 X 10 6 MCF7-pcDNA3 or MCF7-VEGFR-3 cells subcutaneously implanted into the right flank of 6-week old nude mice. Caliper measurements of tumor length and width were used to estimate tumor volume (L X W 2 X 0.5). B.) Table demonstrating tumor incidence, median tumor volume of MCF7-pcDNA3 and MCF7-VEGFR-3 with and without hormonal stimulation. eighteen days after subcutaneous right flank 49

PAGE 50

implantation. Without hormonal stimulation, MCF7-VEGFR-3 formed tumors (93%) with a mean tumor volume of 2153395 mm 3 ; MCF7-pcDNA3 mice failed to develop tumors. With estrogen stimulation both MCF7-pcDNA3 and MCF7-VEGFR-3 cells formed tumors. However, tumors formed by MCF7-VEGFR-3 are ten times larger in volume than those formed by MCF7-pcDNA3 cells (***, p<0.005) C.) Photographs of the median tumors in mice 18 days after implantation are shown and a Western blot analysis of tumor lysate from selected harvested MCF7-pcDNA3 and MCF7-VEGFR-3 xenografts, demonstrating overexpression of highly phosphorylated VEGFR-3 in MCF7-VEGFR-3 xenografts compared to the absence of VEGFR-3 expression in MCF7-xenografts. D.) Imunnohistochemical staining of selected MCF7-pcDNA3 and MCF7-VEGFR-3 xengrafts with LYVE-1 for lymphatic endothelial cells. The staining of LYVE-1 immunoreactive cells in tumor periphery of MCF7-VEGFR-3 xenografts in significantly higher than that of MCF7-pcDNA3 (**p<0.001), indicating increased peritumoral lymphangiogenesis associated with the VEGFR-3 overexpressing MCF7-VEGFR-3 cell xenograft. 50

PAGE 51

CHAPTER 3 THE NOVEL FOCAL ADHESION KINASE INHIBITOR C4 TARGETED TO THE BINDING SITE OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 3 INHIBITS BREAST CANCER PROLIFERATION IN VITRO AND IN VIVO. Abstract Tumor cells survival depends on activation of survival signaling pathways that suppress normal apoptotic stimuli. FAK and VEGFR-3 are tyrosine kinases that have been identified as critical signaling molecules for these host-tumor interactions. We developed a novel FAK-targeted cancer therapy approach by targeting the FAK-VEGFR-3 protein-protein interaction. We have shown previously a direct physical interaction between VEGFR-3 and FAK in cancer cells and we have identified a peptide sequence from the VEGFR-3 binding site which caused apoptosis in cancer but not normal cells. In silico modeling demonstrates that the peptide binding site, localized on the FAT domain of FAK, is an appropriate target for non-peptide small drug-like molecule binding. We used structure-based molecular docking and performed in silico screening of a chemical library of small molecule compounds and identified a small molecule C4 (Chlorpyramine hydrochloride) that is capable of mimicking the peptide that binds to FAK and causes of breast cancer cells. Co-crystalization of the FAT domain of FAK with C4 revealed that binding of C4 occurred in the predicted site. We evaluated C4 for inhibition of FAK-VEGFR-3 binding in the breast cancer cell line BT474 and a model cell line MCF7-VEGFR-3 overexpressing receptor in in vitro and in vivo experiments. We showed the specificity of the selected small molecule in colocalization immunofluorescence experiments, demonstrating that the localization of FAK and VEGFR 3 was changed and the number of FAK-VEGFR-3 complexes was decreased in comparison with unchanged localization of paxillin. In vitro C4 causes dephosphorylation of FAK and VEGFR-3, resulting in inhibition of breast cancer cell 51

PAGE 52

proliferation in a timeand dose-dependent manner with 10 M C4 causing a greater than 50% decrease of proliferation of BT474 and MCF7-VEGFR-3 cells (24 hours), and subsequent dose-dependent induction of apoptosis (48 hours). Inhibition of FAK-VEGFR-3 crosstalk with C4 also inhibited motility and invasion of BT474 and MCF7 model cells. All these effects are more evident in the cells overexpressing VEGFR-3. Moreover, C4 enhanced doxorubicin-mediated growth inhibition by 2 fold in BT474 and MCF7-VEGFR-3 cells. In vivo, FAK and VEGFR-3 inhibition by C4 reduced tumor growth in the BT474 and MCF7-VEGFR-3 models by 80% (all P values <0.01) compared with vehicle treated controls. The effect on tumor growth of C4 (Chlorpyramine hydrochloride), which belongs to the class of histamine receptor H1 blockers, was compared with effect of a different histamine blocker diphenhydramine. We confirmed that the antitumor effect of C4 was not related to its antihistaminergic properties, as diphenhydramine did not cause tumor size reduction in both tumor models. Concomitant administration of C4 with doxorubicin had a great antitumor effect and gave 85% reduction in both tumor models. Our data demonstrate that peptide binding sites are appropriate targets for non-peptide small drug-like molecules and small molecule inhibitors of FAK binding sites can be identified as lead compounds to provide the basis for novel specific cancer therapeutic agents. Introduction The development of invasive and metastatic cancer requires that tumor cells acquire the ability to survive the apoptotic stimuli associated with abnormal proliferative signaling, cell migration and invasion. A number of tyrosine kinases have been linked to tumorigenesis. FAK and VEGFR-3 are tyrosine kinases that have been identified as critical signaling molecules for these host-tumor interactions. 52

PAGE 53

FAK is a protein tyrosine kinase that is localized at contact points between cells and their extracellular matrix (ECM) and is a point of convergence of a number of signaling pathways associated with cell adhesion, invasion, motility, mitogenesis, angiogenesis and oncogenic transformation. This signaling requires both FAK kinase activity and its ability to form multi-protein complexes. Targeting of FAK by anti-FAK antibody, FAK dominant negative FAK-CD, antisense oligonucleotides or siRNA results in cell rounding, detachment, and apoptosis. FAK is emerging as attractive target for the treatment of cancer because it has been shown that FAK is an important survival molecule that is upregulated in a broad range of solid tumors and is expressed at very low levels in normal tissues, creating a therapeutic window for FAK-targeted anticancer therapy FAK is predictive of poor clinical outcome for some types of tumors Indeed, control of FAK signaling has been suggested as a potential anticancer therapy and several FAK inhibitors have recently been developed VEGFR-3 (Flt4) is a receptor tyrosine kinase that plays a major role in lymphangiogenesis and has also been linked to tumorigenesis. VEGFR-3 is activated by its specific ligands, VEGF-C and VEGF-D which promote cancer progression. The VEGF-C/VEGFR-3 axis is expressed in a variety of human tumor cells and activation of VEGF-C/VEGFR3 axis in several tumor types has been shown to promote metastasis(28). The VEGF-C/VEGFR-3 axis plays an important role in cancer cell proliferation, survival and resistance to chemotherapy. Recently, we demonstrated that VEGFR-3 overexpression in absence of ligand expression promotes breast cancer cell survival in vitro and tumor formation in vivo in a xenograft mouse model of human breast cancer. Importantly, it has been shown that inhibition of VEGFR-3 signaling leads to both regression of the lymphatic network and to suppression of tumor lymph node metastasis. So VEGFR-3 is also a validated and attractive target for cancer 53

PAGE 54

therapy. Moreover, FAK is also overexpressed in endothelial cells where VEGFR-3 is mainly represented and inhibition of both of these molecules will likely have dual effect on tumor and its microenvironment. Previously we have shown the physical interaction between VEGFR-3 and FAK in cancer cells and acting along with VEGFR-3 as anchorage-independent survival signals, FAK has distinct functions helping cancer cells resist apoptosis. Herein the FAK-VEGFR-3 interaction is targeted as a means of disrupting the prosurvival multi-protein complex. Protein-protein interactions are very important in determination of the cell path and recent studies of interactions among proteins in the Bcl-2 family have significantly increased our understanding of how interactions determine cell survival or death Especially attractive now seems the approach to target previously undrugable protein-protein interactions with small molecules. The use of small organic molecules as inhibitors of intracellular proteinprotein interactions has been regarded as high risk because of the relatively large surface areas that are often involved in protein-protein pairing But there are an increasing number of successful small molecule drugs inhibiting protein-protein interactions The discovery of small organic molecules that inhibit protein complexes that regulate apoptosis is growing and recently described compounds have helped to characterized Bcl-2, MDM2 and XIAP as drug targets Development of new computational method of analysis and prediction of protein structure, protein-protein interactions and docking of libraries of small molecules is also improved. Molecular docking has led to the successful discovery of novel ligands for more than 50 targets Thus, the FAK-VEGFR-3 interaction provides essential survival signals for cancer cells and targeting this interaction may have a potential role in developing novel molecular therapeutics to treat human tumors. FAK inhibition appears to have minimal effects on non54

PAGE 55

transformed cells. Antisense inhibition of FAK or overexpression of the carboxy-terminal domain FAK-CD, which inhibits FAK function probably by competing for proteins normally associating with the FAK protein, does not induce apoptosis in normal cells. We have identified the peptide sequence from FAK-VEGFR3 binding site and this 12 amino acid peptide decreased proliferation and caused cell detachment and apoptosis in breast cancer cell lines but not in normal breast cells In the current study we utilized the solved crystal structure of the FAK FAT domain for molecular docking of small molecules onto the VEGFR-3 binding site on FAK and functionally evaluated the top scoring drug-like small molecules. The screening identified a compound, C4 (Chlorpyramine hydrochloride), that was functionally equivalent to the FAK-inhibiting peptide from VEGFR-3 binding site. In vitro testing of this compound resulted in the selective induction of apoptosis in many cancer cell lines, especially those overexpressing VEGFR-3. C4 affected proliferation and caused apoptosis in breast cancer cells. In vivo, FAK and VEGFR-3 inhibition by C4 reduced tumor growth and concomitant administration of C4 with doxorubicin had even greater effect. Targeting the FAK-VEGFR-3 interaction represents unique approach to treating cancer because simultaneous FAK and VEGFR-3 inhibition might affect tumor and endothelial cells, resulting in inhibition lymphangiogenesis. Importantly, C4s ability to sensitize breast cancer cells to chemotherapy might permit decreases in dosage, limiting toxicity and improving patients quality of life. Material and Methods Virtual Screening The DOCKv5.2 package was used for in silico screening of 140 000 compounds available from the National Cancer Institute Developmental Therapeutics Program. This computer database was prepared with DOCK accessory software (SF2MOL2, University of California San 55

PAGE 56

Francisco) and Sybyl (Tripos, Inc). Each compound was docked as a rigid body in 100 different orientations. The orientations were filtered by default bump filter parameters to exclude compounds with pronounced steric clashes. The grid-based scoring system was used for scoring with the nonbonded force field energy function implemented in DOCK. A standard 6 to 12 Lennard-Jones potential was used to evaluate van der Waals contacts. Spheres used by DOCK during matching algorithms were generated by SPHGEN. Sites for molecular docking were identified by structural analysis The University of California, San Francisco (UCSF) DOCK suite was used for docking preparation and computation. All heteroatoms and water molecules were removed, and a single chain was isolated in the coordinate file. The program DMS was used to generate a molecular surface (Richards, 1977). SPHGEN was used to generate spheres on the surface of the protein, and a subset of these spheres within 5 of HIS1025 was selected to constrain the search space. Molecular mechanics force field grids were generated using the program GRID, using the standard 6-12 Lennard-Jones function to approximate the van der Waals forces. Finally, DOCK 5.2 was executed using the prepared files and a database of approximately 140,000 small molecules obtained from the National Cancer Institute Developmental Theraputics Program (NCI DTP). We have previously described the preparation of the database (Sandberg, 2005). The top 40 compounds predicted to interact with the target site were subsequently obtained from the NCI. After ranking with DOCK, the top-scoring compounds were tested in vitro. Co-crystallization of C4 and Focal Adhesion Kinase FAT Domain The hanging drop method of vapor diffusion was used in all crystal screens. 1 L of 60 mM C4 dissolved in DMSO was dried on 48 siliconized cover slips for 24 hours. A four L drop consisting of 2 L well solution and 2 L FAK FAT domain at a concentration of 10 mg/mL was placed on the dried cover slip. Initial screen conditions consisted of Crystal Screen 56

PAGE 57

1 from Hampton Research. Tiny crystals were observed within three weeks under condition 45 (0.2M Zinc acetate dihydrate, 0.1M sodium cacodylate trihydrate ph 6.5, 18% w/v polyethylene glycol 8,000). This condition was optimized over a range of pH and (6.2 6.4) and precipitant (20% 25% PEG), and zinc concentration was increased to 0.4M. Maximally sized crystals were obtained in three weeks under these conditions. All crystals were grown at 22 degrees C. X-ray Data Collection and Refinement X-ray data sets were collected at beamline X6A of the National Synchrotron Light Source at Brookhaven National Laboratories in Upton, NY. Data sets were collected for two crystals of FAK FAT domain co-crystallized with compound C4; the better of these data sets scaled to 1.99 and was selected for subsequent refinement. HKL2000 (Otwinowski et al) was used to autoindex and scale the diffraction images. Molecular replacement using MOLREP (Collaborative Computational Project 4) was used to phase the data set, and CNS (Brunger et al) was used for several rounds of refinement. An unbiased composite omit map generated from the data set revealed significant unaccounted-for density in the region of HIS1025, and the structure of C4 was fitted. Additional rounds of refinement added water molecules and further improved the model. Data collection and refinement statistics are listed in Table 1 in Supplemental materials. Cell Lines MCF7, MDA231, T47D breast cancer, A549 lung, SAOS-2 osteosarcoma, A375, C8161 melanoma PANC1 pancreas, HT29 colon cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The BT474 cells are a subclone of the original cell line that does not express the receptor tyrosine kinase Her-2/neu. BT474 were maintained in RPMI-1640 with 10% fetal bovine serum and insulin 250 mcg/ml. MCF7-pcDNA3 and MCF757

PAGE 58

VEGFR-3 stable cell line were produced as described. All cells are maintained in correspondence with ATCC recommendations. All cell lines were incubated at 37 o C in 5% CO 2 Antibodies and Reagents Santa Cruz Biotechnology, Inc (Santa Cruz ,CA, USA): VEGFR-3 C-20 (sc-321) Calbiochem (San Diego, CA, USA): p-VEGFR-3 (pc460) Cell Signaling Technology (Danvers, MA, USA): Pro-caspase-8 (#9746), Erk 1,2 (#9102), p-Erk (#4377S), Akt (#9272), p-Akt (9271S), PARP (#9542) Rabbit Anti-Human VEGF Receptor 2 / 3, phospho PhosphoDetect Polyclonal Antibody FAK 4.47 (Upstate #) C20, Paxillin, phosphor-tyrosin 4G10 (Upstate). Compound C4 Chlorpyramin hydrochloride, NSC 409949 MW: 326.264040 g/mol | MF: C 16 H 21 Cl 2 N 3 Sigma #1915 IUPAC: N-[(4-chlorophenyl)methyl]-N',N'-dimethyl-N-pyridin-2-ylethane-1,2-diamine hydrochloride, Synonym N-p-Chlorobenzyl-N,N-dimethyl-N-(2-pyridyl)ethylenediamine, CAS Number 6170-42-9 Suprastin (solution for injection 20 mg/ml, EGIS (Hungary) 60 mcl/injection. 60 mg/kg. Diphenhydramine (solution for injections 10 mg/ml) 60 mcl / injection. The ED50 of diphenhydramine to be 38 mg/kg. Immunocytochemistry Cells were incubated in presence or absence of C4 and stained with anti-FAK monoclonal antibody 4.47 (Upstate) along or in combination with paxillin () and VEGFR-3 as previously described (43). Detection in case of mono-staining was done with Alexa 546 secondary antibody, and for dual staining combination of Alexa 488 and Alexa 546 secondary antibody was used. 3 X 10 4 cells were plated in 24-well plates with a cover slip placed at the bottom of the well. Upon completion of appropriate treatment course cells on the coverslip were 58

PAGE 59

washed three times with PBS. Cells were fixed by adding 3.7% formaldehyde in PBS for 15 minutes. Cells were washed three times with PBS. Cells were permeabilized with 0.1% Triton-X100 in PBS for 3 minutes. Cells were washed three times with PBS. Cells were blocked with 10% normal goat serum (NGS) in 2%BSA-PBS for 1 hr. Cells were washed three times with PBS. Cells were incubated with primary antibody at a concentration of 5 g/ml in 2% BSA-PBS for 1 hour. Cells were washed three times with PBS. Cells were incubated with the appropriate secondary antibody-florescent molecule conjugate, Alexa-488 (Invitrogen, Carlsbad, CA, USA), diluted 1:100 in 10% 2%BSA-PBS for 45 minutes in the dark. Cells were washed three times with PBS. Cells on cover slip were mounted to slides using Vectashield Hard Set mounting medium (Vector Laboratories, Burlingame, CA, USA).with DAPI. Assays of Cell Viability. Cell survival was assayed by measuring mitochondrial dehydrogenase activity, conversion of soluble MTT into insoluble formasan product as described by Mosmant (29). 5.0 X 10 3 (100 L) cells were plated in 96-well plates and were allowed to attach overnight. One hundred microliters of fresh media with or without C4 was added to each well. Cells were treated for designated amount of time. Each well was incubated with twenty microliters of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) for 1-2 hours. Colorimetric changes were read at wavelength of 490 nm using Benchmark microplate reader (Biorad, Hercules, CA, USA). For each cell line, the concentration-response curve was plotted from which concentrations that produced 50% (EC 50 ) or 25% (EC 25 ) inhibition of cell viability were calculated. Detection of apoptosis was performed by TUNEL assay and Hoechst 33342 staining. Cells were collected by centrifugation, fixed in 3.7% formaldehyde in 1xPBS for 10 min, washed 59

PAGE 60

twice with 1xPBS and stained with Hoechst 33342 or spread on the slide for TUNEL staining. TUNEL assay was done with the APO-DIRECT kit (Pharmingen, BD Biosciences, San Diego, CA) according to the manufacturer's recommendations. Briefly, 1 10 6 cells were fixed with 2% (w/v) electromicroscopic grade paraformaldehyde in PBS on ice for 1 h. After centrifugation, cells were resuspended in ice-cold 70% ethanol and incubated overnight at 20 C for permeabilization. Cells were washed twice with PBS/0.2% BSA, pelleted and incubated at 37 C for 2 h with staining solution containing TdT enzyme and 2-deoxyuridine 5-triphosphate (FITC-dUTP). After two washes with PBS, the pellet was resuspended in a propidium iodide/RNase A solution, incubated for 60 min in the dark, and analyzed by flow cytometry (see above). Quantitative analysis of apoptosis was performed using FlowJo program (Tree Star, Ashland, OR). The percent of apoptotic cells was calculated as the ratio of apoptotic cells to total number of cells counted in several fields with fluorescent microscope in three independent experiments. Simultaneous staining and quantification of apoptotic cells with Hoechst 33342 and TUNEL methods produced very similar results. Western Blot Analysis. Appropriately treated or non-treated cells were allowed to grow until they are 80-85% confluent or until treatment was completed. Cells were twice washed with ice-cold phosphate-buffered saline (PBS), then incubated on ice with 1% NP-40 lysis buffer with inhibitors mM NaCl, 20 mM Tris-Base, pH 7.4, 5 mM EDTA 1% Nonidet P-40, 50mM NaF, 10mM NaVO 4 100 L phosphatase inhibitor cocktail I and II, according to the manufacturers instructions. (Calbiochem, San Diego, CA, USA), 200 L Complete Protease Inhibitor, according to the manufacturers instructions. (Roche Diagnostics, Mannheim, Germany)for 30 minutes. Plates were scraped and lysate was centrifuged at 14,000 rpm for 30 minutes at 4 C. Protein concentration of cell lysate was measured with colorimetric BCA Protein Assay Kit 60

PAGE 61

(Pierce, Rockford, IL, USA) with bovine serum albumin as a standard. The appropriate amount of Laemmli loading buffer was mixed with protein lysate and boiled for 5 minutes at 95 C. 50 micrograms of total protein were loaded and resolved by SDS-PAGE using 7.5%, 10% or 4-20% Tris-HCl gel (Biorad, Hercules, CA, USA). Resolved bands were transferred onto PDVF membrane for two hours at 80 mV at room temperature or at 20 mV overnight at 4C. PVDF membrane was then blocked with 5% BSA in tris-buffered saline tween (TBST, 25mM Tris-HCl, 125 mM NaCl, 0.1% tween) for one hour. The appropriate primary antibody diluted in 5% BSA-TBST (1:1000) was added for two hours at room temperature or overnight at 4 C. Membrane was then washed three times for five minutes each with TBST. The appropriate secondary antibody conjugated with horseradish peroxidase, diluted in 5%BSA-TBST was added for one hour at room temperature. Membranes were washed three times with TBST, then developed using Western Lightning Chemilumnescence Reagent Plus (Perkin Elmer, Boston, MA, USA). Membrane was stripped using Restore Western Blot Stripping Buffer (Pierce, Rockford, IL, USA) for 15 minutes at 37C, then washed three times in TBST for five minutes each wash and reused. BrdU Incorporation Assay BrdU incorporation was performed using BrdU Cell Proliferation Assay, HTS (Calbiochem, San Diego, CA, USA), according to the manufacturers protocol. 2.5 X 10 3 cells were plated into a 96-well plate and allowed to attach overnight. 100 l of fresh growth media or growth media with treatment was added to each well followed by 20 l of BrdU labeling. Cells were incubated for 24 hours. Labeling solution was removed and 200 l of the fixative solution was added and fixation was allowed to incubate for 30 minutes at room temperature. 100 l of anti-BrdU antibody solution was added to each well and incubated for 1 hour at room temperature. Cells were washed three times with Wash Buffer. 100 l of peroxidase Goat Anti61

PAGE 62

Mouse IgG HRP conjugate solution was added and incubated for 30 minutes at room temperature. Cells were washed three times with Wash Buffer. Wells were flooded with dH 2 O. 100 l of Fluorogenic Substrate Working Solution is added to each well and incubated at room temperature for 30 minutes. 100 l of Stop Solution was added to each well. Plates were read by fluorometer at 320 nm / 460nm. Motility and Invasion Assay Protocol was performed using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Bedford, MA, USA). Cells were grown overnight in serum-reduced growth media with 0.5 % FBS. Matrigel chambers were rehydrated for two hours using 500 l of serum-free media. After rehydration, 750 l normal growth media containing 10% FBS was added to the bottom of the well. 5.0 X 10 4 cells in serum-reduced growth media (500 ul) were added to the matrigel chamber or control chamber. In case of C4 treatment increased concentration of the compound was added to both chambes. Chambers were incubated for 24 h in a cell culture incubator, at 37C, 5% CO 2 atmosphere. Non-invading cells were removed with a cotton swab. Diff-Quik solution was added to a 24-well plate. Matrigel chambers were sequentially transferred through a fixative, then two staining solutions, followed by two beakers of double distilled water and were allowed to air dry. Membranes were mounted onto slides using immersion oil and covered with a cover slip. Cells were counted using light microscopy at 40X magnification. RT-PCR Protocol was performed using Cloned AMV First-Strand cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA). 10 M VEGFR-3 primer (proprietary sequence of R&D Systems, RDP-108-025) was combined with 5 g of RNA and 10 M dNTP mix. 8 l of master mix (cDNA synthesis buffer, 0.1 M DTT, RNaseOUT, DEPD-treated water, Cloned AMV 62

PAGE 63

reverse transcriptase) was added to the PCR tube on ice. Reaction was incubated at 50C for 1 hour. Reaction was terminated by incubating at 85C for 5 minutes. cDNA, primers, dNTP, Herculase are mixed. PCR was performed in the following manner: 10 cycles of 95C for 30 seconds, 55C for 30 seconds, 72C for 70 seconds; 30 cycles of 95C for 30 seconds, 55C for 30 seconds, 72C for 80 seconds. Animal Model Subconfluent BT474 and MCF7-VEGFR-3 cells were collected by centrifugation after treatment with EDTA-trypsin. Cells were washed twice in 1X PBS, and then diluted in sterile 1X PBS to a concentration of 1 X 10 6 cells per 100 l. In accordance with the University of Florida IACUC approved protocol E557, 2 X 10 6 cells (200 l) were subcutaneously injected into the right flank of the 6-week old balbc nu/nu mice, 5 in each group. Treatment with compound C4 was started next day after cells injection. Tumor size was measured thrice weekly and volume was calculated using the formula length X width 2 X 0.5. Animals were sacrificed after 21 days of treatment or when tumor size reached protocol end point. Tumor was excised, measured and preserved for protein and RNA preparation and cytochemistry. Animals were housed in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture. Statistical Analysis Data presented are the means and 95% confidence intervals of the three or more experiments. For in vitro and in vivo experiments comparison between groups were made using a two-tailed two-sample Students t test. Differences for which P value was less than 0.05 were considered statistically significant. 63

PAGE 64

Results Site Selection and Computational Docking for High throughout Virtual Screening of Drug-like Compounds to Develop a Specific VEGFR-3-FAK Inhibitor Previously we have demonstrated binding of the C-terminal domain of FAK with VEGFR-3.We have localized the binding using protein epitopes from the VEGFR3 binding site to the FAT domain of FAK. Nuclear magnetic resonance analysis of the FAK FAT domain / VEGFR-3 peptide complex indicated localized chemical shifts of residue HIS1025 on focal adhesion kinase (Prutzman et al., unpublished data). We hypothesized that a small molecule binding to this site could potentially disrupt the FAK-VEGFR-3 interaction in cancer cells, and thereby induce apoptosis. Therefore, the crystal structure of the Focal Adhesion Targeting (FAT) domain of FAK was obtained from the Protein Data Bank (PDB ID: 1K05), and prepared for computational docking (Figure 3-1)A. For high-throughput screening, we utilized a rapid and more economical structure-based approach combining molecular docking in silico with functional testing in the model system of breast cancer cell line BT474. The atomic coordinates for the solution structure of the human FAK FAT domain (PDB code 1qvx) was used in the molecular docking calculations. 140,000 small molecules from the NCIs Development Therapeutics Program were each positioned in the structural pockets defined by crystallography and scored for electrostatic and van der Waals interactions as implemented in DOCK5.2.0 (UCSF). More than 50 compounds with the highest scores were selected for functional testing. All selected compounds were tested in MTT viability assay on BT474 breast cancer cell line at concentration 100 M (data not shown) and compound C4 was selected for its profound inhibitory effect on cell growth (Figure 3-1B). 64

PAGE 65

C4 Decreases Viability of Many Cancer Cell Types at Low Micromolar Concentrations. To test the effect of selected small molecule we assessed its effect on cell viability of different cancer cell types. We found that C4 decreased viability of breast, colon, lung, osteosarcoma, melanoma, pancreatic cancer cells with IC 50 varying between 1-20 (Figure 3-1C). We concluded that this compound had a profound effect on cell viability. Our next step was to determine the mechanism of this effect by confirming that this compound specifically affected FAK and VEGR-3 and their interaction. To address these questions we used a breast cancer cell line high endgogenous VEGFR-3 expression (Supplement figure 1S), which we previously used to identify the FAK-VEGFR-3 interaction We also employed our previously established model system of VEGFR-3 overexpression in MCF7 breast cancer cells (Paper in publication and Figure S1). MCF7 breast cancer cells express very low levels of endogenous VEGFR-3. We stably transfected MCF7 cells with a VEGFR-3 expression plasmid (MCF7-VEGFR-3), and an empty expression vector, pcDNA3.1B (MCF7-pcDNA3). Crystal Structure of Cocrystalized C4 and FAT. FAK FAT-domain protein was co-crystallized with C4 small molecule. The structure was solved at 1.99 angstrom resolution and revealed that C4 was bound to the region of the FAT domain as predicted by molecular docking. Co-crystalization of the FAT domain of FAK with C4 (Chlorpyramine hydrochloride) revealed that binding of C4 occurred in close proximity to amino acids H1025 (Figure 3-1C). C4 Specifically Decreases Viability of Breast Cancer Cells and Sensitize Tumor Cells to Chemotherapy. Viability experiments showed that BT474 cells were highly sensitive to C4 treatment with 1M concentration causing a 40% reduction in viability after 48 h of treatment (Figure 3-2A). We also found that effect of C4 was more pronounced on this breast cancer cell line in 65

PAGE 66

comparison with normal breast epithelial cells MCF10A (Figure 3-9-A). The specificity of small molecule C4 was tested in our model of VEGFR-3 overexpression comparing MCF7-VEGFR-3 cells with the control cell line MCF7-pcDNA3. After 48 h of treatment with incrementally increasing concentrations of C4 cells viability was measured by MTT assay. We found that even a 100 nM concentration of the compound caused differential inhibition of viability comparing control with VEGFR-3-overexpressing MCF7-VEGFR-3 cells (Figure 3-2B). This difference in viability becomes statistically significant at 1M (p<0.01) and at 10 M concentration the difference reaches twofold (P<0.001; Figure 3-2B). This data suggested that C4 specifically inhibits viability of cells expressing VEGFR-3. Additionally, we tested the FAK specificity of C4 using FAK-knockout and FAK-wild type mouse fibroblasts and demonstrated that the FAK knockout fibroblasts were less sensitive to C4 treatment at 10 M for 24 h, showing no affect their viability (Supplemental figure 3-10-B). When we combined C4 treatment with doxorubicin inhibition in breast cancer cells BT474 we found at least an additive effect of these two drugs on cell viability. 1 M concentration of each drug alone decreased viability by approximately 40% but their combination decreased viability by greater than 60% when compared to untreated control (P<0.05; Figure 3-2C). It is important to mention that MCF10A normal breast epithelial cells were not sensitized to doxorubicin by C4 treatment and dual inhibition did not have any additional effects on viability of normal cells (Supplemental Figure 2S-C). These data demonstrate that C4 decreases breast cancer cell viability in a dose-dependent manner and that it displays specificity in its actions to cancer cells that express VEGFR-3. C4 also sensitizes VEGFR-3 expressing breast cancer cells to standard chemotherapeutics. Next, we evaluated the effect of C4 on the FAK-VEGFR-3 interaction. 66

PAGE 67

C4 Treatment Affects Colocalization and Leads to Redestribution of FAK-VEGFR3 Complexes. We used immunofluorescence confocal microscopy to analyze the distribution of endogenous FAK and VEGFR-3 in BT474 cells with and without treatment with small molecule inhibitor C4. As a control we selected paxillin, a focal adhesion protein and well known binding partner of FAK that binds to the FAT domain close to the VEGFR-3 binding site. Cells were dually immunostained for FAK in combination with either VEGFR-3 or paxillin. Confocal microscopy was performed and the degree of colocalization was calculated by scatter plot analysis. In our positive control stained for FAK and paxillin, we found that approximately 90% of FAK and paxillin molecules were colocalized in BT474 cells mainly in focal adhesions (Figure 3-3A). The colocalization of VEGFR-3 with FAK was also high, giving a colocalization rate of 80% which occurred mainly in cytoplasm, as we also have shown previously (Figure 3-3C). These results confirmed that FAK colocalizes with VEGFR-3 and paxillin in cell under normal conditions. Treatment with C4 for 24 hours did not affect the colocalization of FAK and paxillin, evidenced by only a 3% drop in the number of colocalized molecules after treatment (Figure 3-3B). However, C4 treatment resulted in decreased colocalization of FAK and VEGFR-3 in the cytoplasm of BT474 cells with scatter plot analysis showing that colocalization of FAK and VEGFR-3 drops to 41.7% in cells treated with C4 (Figure 3-3D). This 50% drop in colocalized FAK and VEGFR-3 molecules correlates with redistribution of these proteins after C4 treatment, revealed by 3D reconstruction. We found that C4 treatment leads to redistribution of FAK and VEGFR3 inside the cells but does not affect localization of paxillin (Figure 3-10). Thus, our results from immunofluorescence confocal microscopy showed that endogenous FAK and VEGFR-3 colocalize in the cytoplasm, and that small molecule inhibitor C4 specifically targets the FAK67

PAGE 68

VEGFR-3 interaction, leading to redistribution and decreased colocalization of these proteins within cells. It is known that displacement of FAK from focal adhesions leads to its dephosphorylation. Next, we evaluated the effect of C4-induced redistribution on the phosphorylation of both proteins. C4 Causes Dose-dependent Dephosphorylation of VEGFR-3 and Decreases Total Phosphorylation of FAK. Cells were treated with different concentrations of C4 for 48 h and lysates were analyzed for presence of the phosphorylated form of VEGFR-3. We found that 48 h treatment with 1 M C4 leads to partial dephosphorylation at Tyr1063/1068 in activation loop of the kinase domain and 10 M completely dephosphorylated the protein in both MCF7-VEGFR-3 and BT474 cells (Figure 3-4A, B). This effect is dose and time dependent and we concluded that C4 effect is specific for VEGFR-3, because it does not affect phosphorylation of many other tyrosine kinases (data not shown). The same lysates were analyzed with an antibody specific for FAK Y397, but this analysis did not reveal consistent dephosphorylation of FAK at its main autophosphorylation site. However, there are at least 7 more tyrosine phosphorylation sites in FAK and we decided to assess changes in total FAK phosphorylation by immunoprecipitation. Analysis of FAK precipitates with total p-Tyr antibody and found that 10 M treatment with C4 for 48 h caused a decrease in total FAK phosphorylation (Figure 3-4C). These data demonstrate that small molecule interference with the FAK-VEGFR-3 interaction leads to dephosphorylation of both proteins. Next, we evaluated the biological effects of protein redistribution and dephosphorylation caused by our selected small molecule. 68

PAGE 69

C4 causes Timeand Dose-dependent decreases in Proliferation of VEGFR-3-expressing Cells. Viability assay demonstrated inhibitory effect of C4 on breast cancer cell viability. To distinguish between cytostatic or cytotoxic effect of our selected compound, proliferation assays with cell count and BrdU incorporation were performed on MCF7-VEGFR-3 and BT474 cells. First, we used our model of VEGFR-3 overexpression to evaluate the effect of C4 on proliferation. MCF7 cells transfected with control pcDNA 3 vector did not show any decrease in proliferation even after 24 h of treatment with a 10 M concentration of C4 (Figure 5A left). MCF7 cells stably overexpressing VEGFR3 proliferate much faster (more than 3 fold increase in comparison to MCF7-pcDNA3 cells), and 10 M C4 treatment for 24 h reduces proliferation of these VEGFR-3 overexpressing cells more than two fold. We concluded that antiproliferative cytostatic effect of C4 is VEGFR-3 specific, evidenced by the strong affect of C4 on VEGFR-3 overexpressing cells (Figure 5A). In the case of our breast cancer cell line with high endogenous VEGFR-3 expression, treatment of BT474 cells with C4 lead to dose-dependent decreased cell proliferation (Figure 3-5B). Comparing untreated and 10 M treated cultures demonstrated approximately three fold difference in BrdU incorporation (Figure 5C) after 24 h exposure to C4. C4 treatment inhibited proliferation but did not affect cell number with exposures of 24 hours or less. However, after 48 h of treament cell number dropped more than 5 times, which we suspect was related to apoptosis (data not shown). C4 Treatment Leads to Apoptosis. A 48 h exposure of BT474 cells to 10 M C4 induced apoptosis in 64+3% of cells (Figure 3-5C). The ability of C4 to induce apoptosis was concentration-dependent with 1 M C4 causing approximately 10% apoptosis and 10 M treatment leading to 60% of cell death (Figure 3-5D). In parallel we confirmed the effect of C4 on induction of apoptosis by biochemical 69

PAGE 70

analysis using procaspase 8 and PARP cleavage as marker of the initiation of cell death. Figure 3-5E demonstrates that PARP and procaspase 8 were cleaved in C4 concentration dependent manner. Moreover, consistent with our proliferation data, we found that C4-induced apoptosis was related to VEGFR-3 expression. We treated our model cell lines, MCF7-pcDNA3 and MCF7-VEGFR-3, with 10 M C4 for 48 h and measured apoptosis by TUNEL assay. There was a 4-fold increase in apoptotic cell death in the cell line overexpressing VEGFR-3 (18% versus 76 % respectively). We also confirmed the result found in BT474 cells that level of apoptosis was dose-dependent (Figure 5F). Our biochemical experiments revealed that cleavage of PARP and activation of caspase 8 is minimal in control cell line, but increased proportionally to C4 treatment dose in VEGFR3 overexpressing cells (Figure 3-5G). From these experiments we concluded that C4 caused apoptosis in BT474 and MCF7-VEGFR3 breast cancer cells at 48 hours and that C4s ability to induce apoptosis is related to VEGFR-3 overexpression. C4 Specifically Decreases Motility and Invasion of Breast Cancer Cells. Our previous data have shown that overexpression of VEGFR-3 leads to increased motility and invasion of breast cancer cells. We hypothesize that crosstalk between FAK and VEGFR-3 is involved in both processes and, therefore, treatment with our compound-inhibitor of FAK-VEGFR-3 interaction will affect both processes. BT474, MCF7-pcDNA3 and MCF7-VEGFR-3 cells were plated in Boyden chambers and treated for 24 h with gradually increasing concentrations of C4. Cells were stained and counted, BT474 cells treated with increasing concentrations of C4 demonstrate decrease in motility when compared to control at 1M (* p<0.01) and their motility was completely abrogated at 10 M C4 (Figure 3-6A). Invasion was reduced more than 90 % at 1 M concentration of C4 (Figure 6A). MCF7-VEGFR-3 demonstrates a three-fold increase in motility when compared to MCF7-pcDNA3 cells (* 70

PAGE 71

p=0.01) but treatment with increasing concentrations of C4 for 24 h lead to impairment of motility at 10 M (Figure 3-6B). We did not see a significant level of invasion for MCF7-pcDNA3 cells and this level was not affected by treatment with 10 M of C4 (Figure 3-6B). Invasion of MCF7 cells stably overexpressing VEGFR-3 was 2.5 higher than in control cell line and the invasive capacity of these cells was completely abrogated by C4 1 M treatment (Figure 3-6B). C4 Suppresses Tumor Growth In Vivo. To further validate the activity of C4 small molecule we employed a tumor mouse model. MCF7-VEGFR-3 or BT474 breast cancer cell were subcutaneously implanted into the right flank of female nude mice. Intraperitoneal treatment with compound C4 (60 mg/kg) was started the day after cell implantation. After 21 days of treatment, tumor volume of treated mice was significantly smaller than tumor size in vehicle treated group (Figure 7 A, B). For both cell lines our small molecule selected against FAK-VEGFR-3 binding site reduced tumor growth in vivo by 80%. Concomitant administration of C4 (60 mg/kg, daily) with doxorubicin (3 mg/kg, weekly) reduced tumor growth up to 90 % in BT474 cells (data not shown). Our in vitro experiments with dual therpay suggested that we could reduce concentration of each drug and still demonstrate significant antitumor effects. In the next experiment the concentration of doxorubicin was reduced 10-fold (0.3 mg/kg) and its effect on tumor growth was abrogated (Figure 3-7C). Concentration of C4 was reduced to 10 mg/kg which reproducibly produced a 50% reduction in tumor. However, the combination of these drugs at these concentrations caused a more than 80% reduction tumor growth (Figure 3-7C). We concluded that C4 sensitized breast carcinoma cells to standard chemotherapy, allowing for the dramatic reduction in dosage of this cardiotoxic drug. 71

PAGE 72

C4 (Chlorpyramine hydrochloride) belongs to a class of histamine H1 receptor blockers. To evaluate if the effect was C4 was related to its antihistaminergic effects we compared the effect of C4 on xenograft growth to the effect a unselective histamine blocker diphenhydramine. At a dose 30 mg/kg which is just 1.3.times less then EC50 (38 mg/kg) diphenhydramine did not have any effect on tumor growth (Figure 3-7D). At the same time C4 was used at a concentration of 60 mg/kg which is 1.7 times less of EC50 and reduced tumor growth more than 75%. We confirmed that antitumor effect of C4 is not related to it antihistamine properties. These experiments confirmed the high specificity of our selected small molecule C4. Discussion In this study we utilized unique approach to cancer treatment by simultaneously inhibiting two protein-tyrosine kinases FAK and VEGFR-3 through targeting the site of their protein-protein interaction. This study validated our novel structural target (FAK-binding partner binding site), our computational approach for molecular docking and established proof of principle that our novel structure/function based method can identify compounds that have significant biological effect. C4 was functionally equivalent to a FAK-inhibiting peptide (AV3), the sequence of which was derived from VEGFR-3 at its FAK binding site. However, unlike blocking peptides, selected compounds can be used in in vivo models. Virtual molecular docking identified the FAKand VEGFR-3-inhibiting compound C4 (Chlorpyramine hydrochloride) by its ability to interact with the FAT domain of FAK at the VEGFR-3 binding site. This interaction was verified by co-crystallization of the FAK FAT-domain protein with the C4 small molecule. The structure was solved at 1.99 angstrom resolution and revealed that C4 was bound to the same region of the FAT domain as predicted by molecular docking. Our results support the developed high throughput virtual screening approach which previoulsly predicted a few compounds useful for girase and Jak2 inhibition 72

PAGE 73

Studies with peptide inhibitors have already indicated that blockade of specific proteinprotein interactions have therapeutic promise for treating a variety of human cancers Once regarded as intractable targets disrupted by only large macromolecules, protein-protein interactions are now considered viable targets due in large part to the intensive study of p53-MDM2 interaction Compounds selected for protein-binding of the hydrophobic groove of Bcl-2 and Bcl-XL form a group of small molecules and peptides that stimulate apoptosis in cancer cells and tumors. One small molecule from this group, the antagonist GX15-070 (GeminX), has entered clinical trials. In addition to the Bcl-2 family members, the list of successfully inhibited targets include integrins, IL-2, TNFa, iNOS, XIAP. The big advantage of these inhibitors is their high selectivity. The Nutlins inhibitor of p53-MDM2 interaction activated apoptosis in cells expressing wild-type p53 and show a10-20 fold selectivity for cells with active versus mutated p53. Targeting sites of FAK protein-protein interaction represents a novel approach to FAK inhibition. Until now the main approach to targeting FAK was to inhibit the catalytic activity of the tyrosine kinase by interfering with the binding of ATP. Four such inhibitors were reported, two from Novartis, NVP-TAE 226 and NVP-TAC544 and two from Pfizer, PF-573,228 and PF-271. All of them inhibit FAK phosphorylation on Tyr397, but their cellular effects are different. PF228 does not affect cell growth or induce apoptosis, but inhibits migration and focal adhesion turnover. TAE226 inhibits cell growth and in vivo TAE226 significantly reduces tumor burden in ovarian carcinoma and glioma models. PF271, a small molecule FAK kinase inhibitor, can suppress tumor growth in multiple human subcutaneous xenograft models and is undergoing clinical evaluation as anticancer drug. The main problem with small molecules that target the ATP-binding site is their lack of specificity. 73

PAGE 74

Targeting of the VEGF-C/VEGF-D/VEGFR-3 axis in cancer has focused on the inhibition of lymphangiogenesis and lymph node metastasis through neutralization of the paracrine effects of ligand expression on lymphatic endothelia[27]. Targeting VEGF-C or VEGF-D with soluble chimeras of VEGFR-3, VEGF-C/VEGF-D neutralizing antibodies, and siRNA down regulation has been shown to decrease the lymphatic metastasis of xenografts in animal model. Although successful at reducing lymphatic metastasis, these ways of targeting the VEGF-C/VEGF-D/VEGFR-3 axis focus on ligand neutralization and do not address the effects VEGFR-3 overexpression on the malignant tumor phenotype of the primary tumor in the absence of ligand overexpression Importantly, possible side-effects of anti-lymphangiogenesis-based therapies need to be investigated. Data from Chapter 2 show that VEGFR-3 has a significant impact on aggressive tumor characteristics in the absence of ligand overexpression. The impact of highly phosphorylated VEGFR-3 on breast cancer cell proliferation, motility and survival in vitro and tumor formation and growth in vivo underscores the utility of targeting VEGFR-3 in primary tumors as an anti-cancer therapy. Currently, only the tyrosine kinase inhibitor of VEGFR-3 MAZ-51, an indolinone targeted to the ATP-binding site, has been used to specifically inhibit primary tumor VEGFR-3 activation resulting in suppression of rat mammary tumor growth[31]. MAZ51 has not been used in clinical trials. Clinically, broad range tyrosine kinase inhibitors BAY 43-9006 (sorafenib), PTK787/ZK 222584(vatalanib), AZD2171 and CEP-7055 are being used to target the VEGFR family in addition to other receptor tyrosine kinases with varying success. Specific inhibitors of VEGFR-3 are needed to address the unique influences of VEGFR-3 overexpression on human breast cancer. Our small molecule C4 provides the specificity needed to address both 74

PAGE 75

FAK and VEGFR-3 overexpression in tumors without the toxicity associated therapeutic promiscuity. Surprisingly, C4 (Chlorpyramine hydrochloride) belongs to the class of antagonists of histamine receptor HI. This small molecule was analyzed in tumor mouse experiments of Honti and Puntoky in the early 1960s with the working hypothesis that histamine signal transduction might be involved in carcinogenesis, but their results were inconclusive. In our experiments we have shown high specificity of C4 in inhibiting growth of cells overexpressing FAK and VEGFR-3. We demonstrated that treatment with C4 reduce tumor burden by more than 80% in our two breast xenograft mouse models. We also confirmed that antitumor effect of C4 is not related to it antihistamine properties by comparing its antitumor to uneffective diphenhyramine. The other aspect of our findings is related to tumor resistance to standard chemotherapeutics. Chemoresistance in cancer is frequently associated with deregulation of apoptosis, and FAK itself has been directly implicated in facilitating resistance to standard chemotherapeutics. Sensitizing tumors to cytotoxic drugs provides a great advance in cancer treatment since large numbers of tumors are highly resistant or become resistant to currently-used chemotherapies. Concomitant administration of low-dose C4 with low-dose doxorubicin had great efficacy and induced than 85% tumor growth reduction in both tumor models. In summary, the selected small molecule C4 has a strong antitumor effect in cells that overexpress FAK and VEGFR-3 and sensitizes breast cancer xenografts to therapy with conventional chemotherapeutics. This ability of C4 may permit dosage decreases of toxic chemotherapy and might improve patients quality of life. Our data suggest that small molecule inhibitors of FAK-protein interaction can be identified as lead compounds to provide the basis for specific novel cancer therapeutic agents. Such compounds will be valuable experimental 75

PAGE 76

tools for further analyses of FAK function. Furthermore, they might prove useful pharmaceutically to perturb FAK signaling in cancerous cells overexpressing FAK. 76

PAGE 77

Figure 3-1. Site selection for high throughout virtual screening of drug-like compounds to develop small molecule FAK inhibitors. A.) The crystal structure of the focal adhesion targeting (FAT) domain of FAK is shown in cyan and salmon, and residues that undergo shifts upon peptide binding in NMR studies are shown in magenta. The catalytic tyrosine is shown in green. Red spheres indicate the site defined by the program SPHGEN (UCSF) with chemical and geometric features appropriate for specific small molecule binding. Grey bars demarcate the scoring grid utilized to calculate interactions between potential ligands and the targeted structural pocket. B.) Compound C4 N-p-Chlorobenzyl-N,N-dimethyl-N(2pyridyl)ethylenediamine, Molecular Formula C16H20ClN3 HCl, Molecular Weight 326.26, CAS Number 6170-42-9, PubChem Substance ID 24892447 C.) C4 treatment decreases viability of many cancer cell types. MTT assay was performed on breast (BT474, T47D, MDA MB231), colon (HT-29), lung (A549), osteosarcoma (SAOS-2), melanoma (A375, C8161), and pancreatic (PANC1) cancer cell lines. Cells were plated on 96 well plates and allowed to attach for 24 h, then cells were treated with increasing concentrations of C4 for 48-72 h, then analyzed with CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). D.) Electron 77

PAGE 78

density mapping and ribbon diagram of C4 binding site on FAT domain of FAK. Left: 2fo-fc electron density map at 1 for C4 ligand bound to the Focal Adhesion Kinase FAT domain. E.): Calculated interactions between C4 and surrounding residues of FAK FAT domain. Yellow line indicates hydrogen bond between Serine 939 hydroxyl group (donor) and C4 N3 (acceptor). Red lines indicate hydrophobic interactions between C4 and FAK calculated by LIGPLOT (Wallace et al). Note the -stacking interaction between Histidine 1025 and C4 pyridine ring. D. E. Figure 3-1 Continued. 78

PAGE 79

A. B. C. Figure 3-2. C4 specifically decreases viability of VEGFR-3 overexpressing breast cancer lines BT474 and MCF7-VEGFR-3 cells and increases the sensitivity of breast cancer cells to doxorubicin. A, B.) BT474 breast cancer cells with endogenous high VEGFR-3 expression and stable clones of MCF7 breast cancer cells with low VEGFR-3 expression, transfected with vector pcDNA3 or overexpressing VEGFR-3 were plated on 96 well plate and allowed to attach for 24 h, then cells were treated with increasing concentrations of C4 for 48 h and viability was measured in MTT assay. C4 treatment resulted in a dose-dependent decrease in cell viability specific to VEGFR-3 overexpression C.) Doxorubicin was used at two concentrations 10 M and 1 M as 79

PAGE 80

single treatment and in combination with 1 M C4 for 48 h. Viability was measured in MTT assay. Combination of doxorubicin and C4 decreased viability to a greater extent than single agent therapy. All data are average data of 3 independent experiments. marks statistically significant data with P value <0.01, ** P<0.001. For doxorubicin experiment this value was calculated relative to C4 and Doxorubicin. Figure 3-3. Fluorescence immunostaining of endogenous FAK, paxillin, and VEGFR-3 in BT474 cells and scatter plot analysis of colocalization. Cells were dually immunostained for FAK (red) in combination with paxillin (green) or VEGFR-3 (green) using Alexa Fluor 546 and Alexa Fluor 488 secondary antibodies, respectively. Selected samples were treated with 10 M C4 for 24 hrs and colocalization in treated and untreated cells was assessed by immunofluorescent confocal microscopy and scatter plot analysis. A.) Scatter plot shows colocalization of FAK and paxillin in BT474 cells (89.7%). B.) Colocalization is not affected by treatment with small molecule FAK-VEGFR-3 inhibitor C4 (86.1%). C.) FAK and VEGFR-3 are colocalized in BT474 80

PAGE 81

cells (80.5%). D.) Treatment with small molecule C4 causes a two-fold decrease in colocalization of FAK and VEGFR-3 (41.7%). 81

PAGE 82

A. B. P-VEGFR-3 VEGFR-3 GAPDHC4 (M)-0.1 1 10BT474 C. Figure 3-4. C4 treatment causes dose-dependent dephosphorylation of VEGFR-3 and FAK. MCF7-VEGFR-3. A,B.) and BT474 cells after 24 h of treatment with increasing doses of C4 were analyzed for VEGFR-3 phosphorylation. C4 caused dose-dependent dephosphorylation of VEGFR-3. C.) BT474 cells were treated for 24 h with 10 M and 1 M of C4, lysates were immunoprecipitated with FAK antibody 4.47. Western blot was analyzed with anty-phosphotyrosine antibody 4G10 and redeveloped with FAK antibody, demonstrating that C4 treatment decreases total phosphorylation of FAK. 82

PAGE 83

A. MCF7-pcDNA3 MCF7-VEGFR-30200040006000800000.111000.1110Concentration C4 MRFU (320/460 ) ** B. BT474020004000600080001000000.111000.111000.1110 6 h12 h24 h Concentration C4 MRFU (320/460 ) ** ** ** Figure 3-5. C4 causes timeand dose-dependent decrease in proliferation of VEGFR-3 expressing cells. A.) BrduU incorporation assay of MCF7 cells overexpressing VEGFR3 and control cells pcDNA3. Only VEGFR-3 expressing cells demonstrated dose-dependent decreases in proliferation with C4 treatment. B.) Time course of BrdU incorporation in BT474 cells treated with increasing concentrations of C4. C4 treatment caused dose-dependent inhibition of BT474 cell proliferation P<0.05 **P<0.001. C4 treatment causes apoptosis in BT474 cells. 83

PAGE 84

Figure 3-5. C.) BT474 cells were treated with 10 M C4 for 24 and 48 h and apoptosis was measured by TUNEL assay. C4 does not induce apoptosis at 24 hours, but induces massive apoptosis at 48 hours D,E.) 48 h exposure to C4 leads to dose-dependent cell death by apoptosis and accompanied by PARP degradation and procaspase 8 activation. Data of three independent experiments. F,G.) MCF7 stable clones transfected with pcDNA3 vector or VEGFR-3 expressing plasmid were treated with 10 M C4 for 48 h and apoptosis was measured by TUNEL assay. 48 h exposure to C4 leads to cell death by apoptosis, which is C4 dose-dependent and accompanied by PARP degradation and ProCaspase8 activation. Cells overexpressing VEGFR-3 are more sensitive to apoptosis induced by C4. 84

PAGE 85

F. 020406080100MCF7-pcDNA3MCF7-VEGFR-3Concentration C4 10 M% apoptosis (TUNE L G. Caspase-8PARPC4 (M) -0.1 1 10 -0.1 1 10MCF7-pcDNA3MCF7-VEGFR3 Figure 3-5. Continued. 85

PAGE 86

A. BT47405010015020025030035000.1110Concentration C4 MCell number Motililty Invasion B. 05010015020025030035000.111000.1110 MCF7-pcDNA3MCF7-VEGFR-3 Concentration C4 MCell number Motililty Invasion Figure 3-6. C4 specifically decreases motility and invasion of breast cancer cells in a dose-dependent manner. 5 X 10 4 cells plated on membrane with 8-micron pores with and without matrigel coating in a modified Boyden chamber. Number of cells able to traverse the membrane were measured by cell count after staining. A.) BT474 cells treated with increasing concentrations of C4 demonstrate dose-dependent decreases in motility when compared to control (* p<0.01). Motility is completely abrogated at 10 M C4, but invasion is reduced more than 90 % at 1 M concentration of C4. B.) MCF7-VEGFR-3 demonstrates a three-fold increase in motility when compared to MCF7-pcDNA3 cells (* p=0.01) but treatment with increasing concentrations of C4 for 24 h lead to dose-dependent impairment of motility and invasion at 10 M at 1 M concentrations of C4, respectively. 86

PAGE 87

A. BT47405001000150020002500300013591114172022Days of treatmentTumor Volume (mm3) Vehicle C4 60 mg/kg B. MCF7-VEGFR-305001000150020002500300013591114172022Days of treatmentTumor Volume (mm3) Vehicle C4 60 mg/kg Figure 3-7. C4 reduces tumor growth in xenotransplant mouse model. A, B.) 2 X 10 6 BT474 MCF7-VEGFR-3 cells were subcutaneously inoculated into right flank of mice, 5 mice per group. Treatment with 60 mg/kg C4 or vehicle (PBS) started the day after implantation as daily intraperitoneal (IP) injections Caliper measurements of tumor length and width were used to estimate tumor volume (L X W 2 X 0.5). Mice were sacrificed 21 days after initiation of treatment and tumors were measured for size and weight. The volume of C4-treated BT474 and MCF7-VEGFR-3 xenografts were significantly smaller than vehicle treated tumors from starting at day 14 (* P<0.01). C.) C4 sensitizes tumors to chemotherapy treatment with doxorubicin at very low concentrations. 2 X 10 6 BT474 were subcutaneously inoculated into right flank of 87

PAGE 88

mice. Treatment with 10 mg/kg C4, IP, once a day, doxorubicin 0.3 mg/kg once a week or combination started the day after implantation. Experiment was terminated after 14 days when tumor size of single treatment reached protocol end point. Statistically significant difference (* P<0.01) with vehicle treated tumors registered from day 9. Doxorubicin at chosen low concentration 0.3 mg/kg (3 mg/kg caused approximately 60% reduction of tumor growth) does not affect tumor growth and C4 at 10 mg/kg leads to 50% tumor growth reduction, but combination produced a strong 85% reduction of tumor growth. D.) C4 effect is independent of its antihistaminergic effects. 2 X 10 6 MCF7-VEGFR-3 cells were subcutaneously inoculated into right flank of mice. Treatment with 60 mg/kg C4 (chlopyramine hydrochloride histamine receptor H1 antagonist), 30 mg/kg diphenhydramine (histamine receptor H1 antagonist ) or vehicle (PBS) started next day after inoculation as IP once a day. There was no difference in tumor growth of PBS-treated and diphenhydramine-treated mice. 88

PAGE 89

C. BT474 05001000150020002500300013691114Days of treatmentTumor Volume (mm3) Vehicle Dox 0.3 mg/kg C4 10 mg/kg C4+Dox D. MCF7-VEGFR-305001000150020002500300013681013151821Days of treatmentTumor Volume (mm3) Vehicle C4 60 mg/kg diphenhydramine 30 mg/kg * Figure 3-7. Continued. 89

PAGE 90

Figure 3-8. S. VEGFR-3 Expression in Breast Cancer Cell Lines. A.) Reverse Transcription followed by polymerase Chain Reaction (RT-PCR) was performed with VEGFR-3-specific primers to demonstrate transcription of the VEGFR-3 gene in stably transfected MCF7 cells. BT474 cells also demonstrate VEGFR-3 gene transcription. B.) Western blot analysis demonstrating expression of the VEGFR-3 in stably transfected MCF7 cells. Both BT474 and MCF7-VEGFR-3 cells expresses highly phosphorylated VEGFR-3. Neither the parental cell line nor MCF7-pcDNA3 demonstrate VEGFR-3 transcription or expression. 90

PAGE 91

A. 00.20.40.60.811.200.1110100viability (treated/untreated)Concentration C4 M MCF10A B. C. 00.20.40.60.811.2contC4+DOXDOXC4viability MCF7 MCF10A 91

PAGE 92

Figure 3-9. Specificity of the effect of small molecule C4. A.) Effect of C4 is stronger in cancer cells. Breast carcinoma cell line BT474 and normal breast epithelial cell line MCF10A were treated with increased concentration of C4 for 24 h and viability was measured in MTS assay. BT474 cells significantly (* marked P<0.05 and ** corresponds to P<0.01) more sensitive to C4 concentrations between 0.1 and 10 M, but 100 M concentration is toxic for both cell lines. B.) FAK-dependent effect of C4. Phase contrast picture of the FAK knockout and wild type mouse fibroblasts treated with 10 M C4 for 24 h. Only FAK+/+ cells viability and proliferation is affected after 24 h.treatment with C4. C.) Cancer cells are more sensitive to treatment with compound C4 than chemotherapy drug doxorubicin. Breast carcinoma cell line MCF7 and normal breast epithelial cell line MCF10A were treated with 1 M of C4, 1 M of Doxorubicin or combination for 24 h and viability was measured in MTS assay. MCF7 cells are more sensitive to 1 M C4 than normal cells MCF10A and dual inhibition decreased viability more than 40%. MCF10A normal cells are less sensitive to C4, but doxorubicin alone dramatically decrease their viability more than 40% and dual treatment does not have any additional effect on viability of MCF10A cells. 92

PAGE 93

Figure 3-10. C4 treatment changes localization of endogenous FAK and VEGFR-3 in BT474 cells, but does not affect distribution of paxillin. Confocal images of BT474 cells stained for FAK (left panel), VEGFR-3 (middle panel), and paxillin (right panel) are shown with single XY, XZ, YZ cross-sections through different areas of the cells as indicated by cross-hair lines. We used immunofluorescence confocal microscopy to analyze distribution of endogenous FAK, VEGFR-3, and paxillin in BT474 cells and how it is affected by treatment with small molecule FAK inhibitor C4. As a control we selected paxillin focal adhesion protein, well known binding partner of FAK, bound to FAT domain as VEGFR-3 binds. BT474 breast cancer cells were grown on cover slips and were treated with 10M C4 for 24 hr. Treated and untreated cells were individually immunostained for FAK, VEGFR-3, and paxillin and evaluated by confocal microscopy, followed by 3-D reconstruction. We found that FAK and paxillin stain at focal adhesion sites with some staining throughout the cytoplasm, while VEGFR-3 is localized mainly in the cytoplasm as well as in and around the nucleus (Figure 3A ). Our results indicate that treatment of cells with 10M C4 for 24 hr partially displaces FAK from the focal adhesions and leads to FAK relocalization closer to the cell nucleus (Figure 3A left ), while paxillin cellular distribution remains unaffected by treatment (Figure 3A right). We also found that C4 treatment of BT474 cells results in VEGFR-3 relocalizaion from cellular cytoplasm to perinuclear compartment and to the nucleus (Figure 3A middle panel). Thus, we confirmed that FAK and VEGFR-3 are located in the cytoplasm, and that treatment with C4 specifically affects FAK and VEGFR-3 distribution throughout the cell and leads to relocalization of these proteins, but does not affect paxillin distribution. It is important to note that same redistribution of FAK and VEGFR-3 was documented when BT474 cells were treated with peptide AV3 from the VEGFR-3 binding region (Garces) (that leads us to suggestion that compound C4 has the same effect as AV3 peptide). 93

PAGE 94

A. B. C. BT47400.511.522.53VehicleC4 60 mg/kgWeight (g) MCF7-VEGFR-300.511.522.53VehicleC4 60 mg/kgWeight (g) Figure 3-11. C4 reduced tumor growth in xenotransplant mouse model and sensitize tumors to chemotherapy treatment with doxorubicin. A, B.) Photographs of the median tumors in mice 22 days after subcutaneous right flank inoculation of 2 X 10 6 BT474 (A) or 2 X 10 6 MCF7-VEGFR-3 (B) cells. Treatment with 60 mg/kg C4, IP, once a day started next day after inoculation. Mice were sacrificed 21 days later and tumors were measured for size and weight. C. BT474 and MCF7-VEGFR-3 C4-treated tumors weight in comparison with vehicle treated tumors, P<0.05 94

PAGE 95

Table 3-1. X-ray data collection and refinement statistics Data Collection Space group P2 1 2 1 21 Cell dimensions a = 48.246, b = 50.289, c = 49.532, = = = 90 Wavelength () 0.9322 Resolution range () 30.0-1.99 (2.01-1.99) Completeness (%) 100.0 (100.0) Average I/ 21.9 (3.5) Unique Reflections 8673 (213) Redundancy 5.9 (5.9) Refinement Resolution range () 30.0-1.99 Reflections used in refinement 8625 R work / R free (%) 24.2 / 24.9 # FAT / Asymmetric Unit 1 Mean B-factor ( 2 ) 25.86 Ramachandran Plot Statistics Residues in most favored regions 115 (99.1%) Residues in additionally favored regions 1 (0.9%) Residues in generously allowed regions 0 (0.0%) Residues in disallowed regions 0 (0.0%) Note: Values in parentheses are for the highest resolution shell 95

PAGE 96

LIST OF REFERENCES 1. Barron JJ, et al. Assessing the economic burden of breast cancer in a US managed care population. Breast Cancer Res Treat 2008;109:367-77. 2. Hanahan D and Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70. 3. Schaller MD et al. pp125fak a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proceedings of the National Academy of Sciences of the United States of America 1992;89:5192-6. 4. Kanner SB et al. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A 1990;87:3328-32. 5. Golubovskaya VM, Finch R, and Cance WG. Direct interaction of the N-terminal domain of focal adhesion kinase with the N-terminal transactivation domain of p53. J Biol Chem 2005;280:25008-21. 6. Sieg DJ, et al. FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2000; 2:249-256. 7. Kurenova E, et al. Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein. Mol Cell Biol 2004;24:4361-71. 8. Schaller MD, et al. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 1994;14:1680-8. 9. Han DC and Guan JL. Association of focal adhesion kinase with Grb7 and its role in cell migration. J Biol Chem 1999;274:24425-30. 10. Hildebrand JD, Schaller MD, and Parsons JT. Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. Journal of Cell Biology 1993;123:993-1005. 11. Mortier E, et al. The focal adhesion targeting sequence is the major inhibitory moiety of Fak-related non-kinase. Cell Signal 2001;13:901-9. 12. Renshaw MW, Price LS, and Schwartz MA. Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase. J Cell Biol 1999;147:611-8. 13. Kornberg LJ, et al. Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins. Proc Natl Acad Sci U S A 1991;88:8392-6. 14. Kornberg L, et al. Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase. J Biol Chem 1999;267:23439-42. 96

PAGE 97

15. Burridge K, Turner CE, and Romer LH, Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol 1992;119:893-903. 16. Frisch SM, et al. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 1996;134:793-9. 17. Hungerford JE, et al., Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J Cell Biol 1996;135:1383-90. 18. Ilic D, et al. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol 1998;143:547-60. 19. Cary LA, Chang JF, Guan JL, Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci 1996;109:787-94. 20. Cary, LA, et al. Identification of p130Cas as a mediator of focal adhesion kinase-promoted cell migration. J Cell Biol 1998;140:211-21. 21. Sieg DJ, Hauck CR and Schlaepfer DD, Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J Cell Sci 1999;112:2677-91. 22. Sieg DJ, et al. FAK integrates growth-factor and integrin signals to promote cell migration. Nat Cell Biol 2000;2:249-56. 23. Reiske HR, et al. Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration. J Biol Chem 1999;274:12361-6. 24. Chen SY, Chen HC. Direct interaction of focal adhesion kinase (FAK) with Met is required for FAK to promote hepatocyte growth factor-induced cell invasion. Mol Cell Biol 2006;26:5155-67. 25. Ilic D, et al. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 1995;377:539-44. 26. Klemke RL, et al. CAS/Crk coupling serves as a "molecular switch" for induction of cell migration. J Cell Biol 1998;140:961-72. 27. Tamura M, et al. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 1998;280:1614-7. 28. Abbi S, et al. Regulation of focal adhesion kinase by a novel protein inhibitor FIP200. Mol Biol Cell 2002;13:3178-91. 29. Slack-Davis JK, et al. Cellular characterization of a novel focal adhesion kinase inhibitor. J Biol Chem 2007;282:14845-52. 97

PAGE 98

30. Xu LH, et al., Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells. Cell Growth Differ 1996;7:413-8. 31. Xu LH, et al., The COOH-terminal domain of the focal adhesion kinase induces loss of adhesion and cell death in human tumor cells. Cell Growth Differ, 1998; 9:999-1005. 32. Xu, LH, et al. The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways. J Biol Chem 2000;275:30597-604. 33. Golubovskaya VM, et al. p53 regulates FAK expression in human tumor cells. Mol Carcinog 2008;47:373-82. 34. Zhai J, et al., Direct interaction of focal adhesion kinase with p190RhoGEF. J Biol Chem 2003;278:24865-73. 35. Schmitz KJ, et al. High expression of focal adhesion kinase (p125FAK) in node-negative breast cancer is related to overexpression of HER-2/neu and activated Akt kinase but does not predict outcome. Breast Cancer Res 2005;7:194-203. 36. Nguyen DH, et al. Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner. J Cell Biol 1999;146:149-64. 37. Jo M, et al. Cooperativity between the Ras-ERK and Rho-Rho kinase pathways in urokinase-type plasminogen activator-stimulated cell migration. J Biol Chem 2002;277: 12479-85. 38. Klemke RL, et al. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 1997;137:481-92. 39. Lahlou H, et al., Mammary epithelial-specific disruption of the focal adhesion kinase blocks mammary tumor progression. Proc Natl Acad Sci U S A 2007;104:20302-7. 40. van Nimwegen MJ, et al. Requirement for focal adhesion kinase in the early phase of mammary adenocarcinoma lung metastasis formation. Cancer Res 2005;65:4698-06. 41. Mitra SK, et al. Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model. Oncogene 2006;25:4429-40. 42. Owens LV, et al., Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 1995;55:2752-5. 43. Cance WG, et al. Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clin Cancer Res 2000;6:2417-23. 98

PAGE 99

44. Madan R, et al., Focal adhesion proteins as markers of malignant transformation and prognostic indicators in breast carcinoma. Hum Pathol 2006;37:9-15. 45. Oktay MH, et al. Focal adhesion kinase as a marker of malignant phenotype in breast and cervical carcinomas. Hum Pathol 2003;34:240-5. 46. Watermann DO, et al., Specific induction of pp125 focal adhesion kinase in human breast cancer. Br J Cancer 2005;93:694-8. 47. Lark AL, et al. High focal adhesion kinase expression in invasive breast carcinomas is associated with an aggressive phenotype. Mod Pathol 2005;18:1289-94. 48. Lightfoot HM, et al. Upregulation of focal adhesion kinase (FAK) expression in ductal carcinoma in situ (DCIS) is an early event in breast tumorigenesis. Breast Cancer Res Treat 2004;88:109-16. 49. Planas-Silva MD, et al., Role of c-Src and focal adhesion kinase in progression and metastasis of estrogen receptor-positive breast cancer. Biochem Biophys Res Commun 2006;341:73-81. 50. Wilks AF, et al. The application of the polymerase chain reaction to cloning members of the protein tyrosine kinase family. Gene 1989;85:67-74. 51. Galland F, et al. Chromosomal localization of FLT4, a novel receptor-type tyrosine kinase gene. Genomics 1992;13:475-8. 52. Aprelikova O, et al., FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter. Cancer Res 1992;52:746-8. 53. Pajusola K, et al. FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines. Cancer Res 1992;52: 5738-43. 54. Salameh A, et al., Direct recruitment of CRK and GRB2 to VEGFR-3 induces proliferation, migration, and survival of endothelial cells through the activation of ERK, AKT, and JNK pathways. Blood 2005;106:3423-31. 55. Pajusola K, et al. Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxy terminal tails are produced by alternative processing of primary transcripts. Oncogene 1993;8:2931-7. 56. Pajusola K, et al. Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors. Oncogene 1994;9:3545-55. 57. Fournier E, et al. Mutation at tyrosine residue 1337 abrogates ligand-dependent transforming capacity of the FLT4 receptor. Oncogene 1995;11:921-31. 99

PAGE 100

58. Fournier E, et al. Role of tyrosine residues and protein interaction domains of SHC adaptor in VEGF receptor 3 signaling. Oncogene 1999;18:507-14. 59. Wang JF, Zhang XF, Groopman JE. Stimulation of beta 1 integrin induces tyrosine phosphorylation of vascular endothelial growth factor receptor-3 and modulates cell migration. J Biol Chem 2001; 276:41950-7. 60. Zhang X, Groopman JE, Wang JF. Extracellular matrix regulates endothelial functions through interaction of VEGFR-3 and integrin alpha5beta1. J Cell Physiol 2005;202:205-14. 61. Borg JP, et al. Biochemical characterization of two isoforms of FLT4, a VEGF receptor-related tyrosine kinase. Oncogene 1995;10:973-84. 62. Joukov V, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. Embo J 1996;15:1751. 63. Achen MG, et al. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc Natl Acad Sci U S A 1998;95:548-53. 64. Stacker SA, et al. Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J Biol Chem 1999;274:32127-36. 65. McColl BK, et al. Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D. J Exp Med 2003;198:863-8. 66. McColl BK, et al. Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2. Faseb J 2007;21:1088-98. 67. Joukov V, et al. Proteolytic processing regulates receptor specificity and activity of VEGF-C. Embo J 1997;16:3898-911. 68. Marconcini L, et al. c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro. Proc Natl Acad Sci U S A 1999;96:9671-6. 69. Baldwin ME, et al. Vascular endothelial growth factor D is dispensable for development of the lymphatic system. Mol Cell Biol 2005;25:2441-9. 70. Makinen T, et al. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. Embo J 2001;20:4762-73. 71. Makinen T, et al. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat Med 2001;7:199-205. 100

PAGE 101

72. Kaipainen A, et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A 1995;92:3566-70. 73. Kukk E' et al. VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development 1996;122:3829-37. 74. Veikkola T, et al. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. Embo J 2001;20:1223-31. 75. Pytowski B, et al. Complete and specific inhibition of adult lymphatic regeneration by a novel VEGFR-3 neutralizing antibody. J Natl Cancer Inst 2005;97:14-21. 76. Irrthum A, et al. Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase. Am J Hum Genet 2000;67:295-301. 77. Karkkainen MJ, et al. Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat Genet 2000;25:153-9. 78. Persaud K, et al. Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C. J Cell Sci 2004;117:2745-56. 79. Dumont DJ, et al. Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 1998;282:946-9. 80. Akahane M, et al. Vascular endothelial growth factor-D is a survival factor for human breast carcinoma cells. Int J Cancer 2006;118:841-9. 81. Timoshenko AV, Rastogi S, Lala PK. Migration-promoting role of VEGF-C and VEGF-C binding receptors in human breast cancer cells. Br J Cancer 2007;97:1090-8. 82. Garces CA, et al. Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells. Cancer Res 2006;66:1446-54. 83. Roberts N, et al. Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2. Cancer Res 2006;66:2650-7. 84. Gunningham SP, et al. The short form of the alternatively spliced flt-4 but not its ligand vascular endothelial growth factor C is related to lymph node metastasis in human breast cancers. Clin Cancer Res 2000;6:4278-86. 85. Bando H, et al., The association between vascular endothelial growth factor-C, its corresponding receptor, VEGFR-3, and prognosis in primary breast cancer: a study with 193 cases. Oncol Rep 2006;15:653-9. 101

PAGE 102

86. Jacquemier J, et al. Prognosis of breast-carcinoma lymphagenesis evaluated by immunohistochemical investigation of vascular-endothelial-growth-factor receptor 3. Int J Cancer 2000;89:69-73. 87. Nakamura Y, et al. Clinicopathological significance of vascular endothelial growth factor-C in breast carcinoma with long-term follow-up. Mod Pathol 2003;16:309-14. 88. Mylona E, et al. Clinicopathological and prognostic significance of vascular endothelial growth factors (VEGF)-C and -D and VEGF receptor 3 in invasive breast carcinoma. Eur J Surg Oncol 2007;33:294-300. 89. Akahane M, et al. A potential role for vascular endothelial growth factor-D as an autocrine growth factor for human breast carcinoma cells. Anticancer Res 2005;25:701-7. 90. Karpanen T, et al. Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res 2001;61:1786-90. 91. Mattila MM, et al. VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors. Int J Cancer 2002;98:946-51. 92. Skobe M, et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001;7:192-8. 93. Kinoshita J, et al. Clinical significance of PEA3 in human breast cancer. Surgery 2002;131:S222-5. 94. Schoppmann SF, et al. VEGF-C expressing tumor-associated macrophages in lymph node positive breast cancer: impact on lymphangiogenesis and survival. Surgery 2006;139:839-46. 95. Laakkonen P, et al. Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer Res 2007;67:593-9. 96. Valtola R, et al. VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am J Pathol 1999;154:1381-90. 97. Longatto-Filho A., et al. VEGFR-3 expression in breast cancer tissue is not restricted to lymphatic vessels. Pathol Res Pract 2005;201:93-9. 102

PAGE 103

BIOGRAPHICAL SKETCH Darrell Lamont Hunt was born to John Wesley and Mary Ann Hunt in 1976 in Miami, Florida. Darrell attended Miami Central Senior High School where he graduated as the class of 1994 valedictorian. He left Florida to attend the University of North Carolina at Chapel Hill where graduated with high honors, receiving a Bachelor of Science in chemistry in 1998. Darrell attended medical school at the Washington University of School of Medicine in St. Louis, MO where he received a Medicinae Doctoris in 2002. Dr. Hunt completed two years residency training in general surgery at the University of Florida College of Medicine before joining the Interdisciplinary Program in Biomedical Sciences to pursue a Doctor of Philosophy in medical sciences, studying protein-protein interactions in the promotion of breast cancer cell growth, survival, and metastasis. Upon completion of these studies he will return to the University of Florida School of Medicine to complete a residency training program in general surgery, followed by a fellowship in surgical oncology.