ANGIOTENSIN-(1-7): A NOVEL SMALL MOLECULE FOR THE TARGETED TREATMENT OF TRIPLE NEGATIVE BREAST CANCER

BY

DAVID RICARDO SOTO-PANTOJA

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY

GRADUATE SCHOOL OF ARTS AND SCIENCES

In Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

In Molecular Genetics & Genomics

DECEMBER 2008

Winston-Salem, North Carolina

Approved By: Dr. E. Ann Tallant, Ph.D., Advisor

Examining Committee: Dr. Patricia E. Gallagher, Ph.D, Chair Dr. Suzy Torti, Ph.D. Dr. Linda Metheny-Barlow, Ph.D. Dr. Donald Bowden, Ph.D.

ii

I dedicate this work to my mother Adelina Pantoja to whom I owe everything and

my godparents Gullermo and Carmen Vila for their unconditional support.

iii

AKNOWLEDGEMENTS

A lot of people have gone further than they thought they could because

someone else thought they could. ~ Unknown. I was lucky enough to find two

people who saw my potential and never gave up on my goal of becoming a

scientist, my mentors Dr. Ann Tallant and Dr. Patricia Gallagher. Thank you for

the enthusiasm, inspiration and patience you have given me. I would not have

accomplished this project without your encouragement and sound advice; I will

always be indebted to both of you for your careful training that not only has

developed my scientific principles but help me grow as a person.

I want to express my sincere thanks to my committee members, Dr. Suzy

Torti, Dr. Linda Metheny-Barlow and Dr. Donald Bowden. In spite of their busy

schedules, they have all made themselves available and provided insight into my

project from an outside perspective.

I am very much obliged to Dr. Debra Diz, who originally gave me the

opportunity to come to Wake Forest as a member of the Post-Baccalaureate

Research program. Through her guidance and support I found a niche in which I

could thrive and show my true potential as a researcher. I am grateful for the

support of Dr. Mildred Chaparro my undergraduate research advisor who guided

and motivated me to continue my graduate studies. I would like to thank the

Faculty of the Hypertension and Vascular Research Center for their endeavor of

making this department an exceptional training environment. I would also like to

iv

thank the administrative staff of the Hypertension Center, especially Tina Williard

for looking after me when I first arrived to Wake. I would also like to thank Dr.

Mark Lively and the Molecular Genetics Program members for their support

during my years as a graduate student at Wake Forest. I am thankful to Ms.

Susan Pierce, and the Graduate School Administrative Staff who rendered all the

administrative support and let me focus on the science.

It is difficult to overstate my gratitude to the Tallant-Gallagher laboratory

Staff: my friends Robert Lanning, Tenille Howard, Mark Landrum and past

members Brian Bernish and Randi Leonard. I will always appreciate your

patience to teach me new lab skills especially in the early stages of my training; I

thank you for interrupting your own work to give your time, help and knowledge to

troubleshoot my own experiments. Thank you for making the lab the wonderful

working environment that it is; I will always cherish our conversations, jokes and

laughter.

I have no words that can express my gratitude and my feelings towards

my colleagues, Jyotsana Menon and LaTronya McCollum. We will always share

a special bond since we started this journey together in our lab. Thank you for

your assistance with my experiments but more for your words of encouragement

and warm friendship. I will never forget our ingenious ways to solve problems

together. I would also like to thank past students Kristin Dickson and Steven

Newton for their help and for their wonderful friendship. I would like to credit Alex

v

Pirro for pursuing his interest in science through the assistance with my

experiments. I want to acknowledge newer members of the lab Kyle Carver and

Bhavani Krishnan for their support in laboratory activities and their comradeship.

I have no words to thank Katherine Cook for her support and insight, on our

projects that relate to the treatment and improvement of quality of life for breast

cancer patients with Ang-(1-7) treatment; even though you came at a later point

in my graduate career, you have made an enormous impact on my career and in

my personal life. I will always be grateful.

I would like to express my deep sense of gratitude to my friends, Shea

Gilliam-Davis, Exazevia Logan, Karl Pendergrass, and Amy Arnold without

whose unconditional support and constant encouragement, the successful

completion of this research work would not have been possible. I would also like

to thank my friend Aaron Trask for the many hours that we spent together

studying in our initial years, his scientific insight, the stress-relieving racquetball

games and the many times we fixed cars and household items.

I would like to thank my postdoctoral friends especially Dr. Maria Garcia-

Espinoza for helping me to adjust when I arrived to Winston-Salem, for all your

long lasting support, advice and friendship I will always be indebted to you. I

would like to acknowledge the long-standing support of my network of

undergraduate friends Drs Karla Rodriguez, Nadya Morales, Edgardo Sanabria,

Katheleen Rivera, Yeissa Chabrier, Eliany Sanchez and Orlando Marrero. Your

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warm friendship, affectionate encouragement and unfailing response has allowed

me continue and finish successfully my work as a graduate student.

Last but no least I would like to thank my family--my sister Frances and

my brother Hector and my cousins Sharon and Nadya Vila--for their support and

for always believing I could achieve any goals I proposed my self to. Again I

would like to reiterate my gratefulness to my Mother and My Godparents to

whom I have dedicated this work; without their love and support I could have not

completed these studies successfully. Above all, I thank the God for the

blessings and for placing all these people in my path who have helped me in

accomplishing this task.

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TABLE OF CONTENTS

Page List of Figures viii List of Tables xi List of Abbreviations xii Abstract xvi Chapter I Introduction 1 Chapter II Materials and Methods 64 Chapter III Results 88 Chapter IV Discussion 168 References 190 Scholastic vitae 208

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LIST OF FIGURES

CHAPTER I Page

Figure 1 The renin-angiotensin system 4

Figure 2 Chemical structure of Ang-(1-7) 7

Figure 3 Pathways for the formation of Ang-(1-7) and effect of ACE inhibition on Ang-(1-7) circulating levels 11

Figure 4 In vitro and in vivo anti-proliferative effects of Ang-(1-7) 18

Figure 5 Inhibition of human lung tumor xenograft by Ang-(1-7) 24

Figure 6 Molecular profiling of breast tumors 31

Figure 7 Tumor angiogenesis 46

Figure 8 Mitogen-activated protein kinase pathways 59

CHAPTER III

Figure 9 Effect of Ang-(1-7) on cell number 90

Figure 10 Inhibition of orthotopic breast tumor growth by Ang-(1-7) 95

Figure 11 Reduction in tumor weight by Ang-(1-7) 97

Figure 12 Effect of Ang-(1-7) on tumor cell proliferation 99

Figure 13 Effect of Ang-(1-7) on COX-2 gene expression in triple negative orthotopic tumors 102

Figure 14 Effect of Ang-(1-7) on PGES gene expression in

triple-negative orthotopic tumors 104

Figure 15 Effect of Ang-(1-7) on MAP kinase activity in breast orthotopic tumors 108

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Figure 16 Effect of Ang-(1-7) on MEK1/2 activity in breast orthotopic tumors 110

Figure 17 Effect of Ang-(1-7) on DUSP1 activity in

MDA-MB-231 orthotopic breast tumors 114

Figure 18 The up-regulation of DUSP1 by Ang-(1-7) is time-dependent 116

Figure 19 Ang-(1-7) up-regulates DUSP1 protein expression through an AT(1-7) receptor 118

Figure 20 Ang-(1-7) reduces tumor vessel density in TNBC tumors 122

Figure 21 Effect of Ang-(1-7) on VEGF protein and gene expression in triple-negative orthotopic tumors 124

Figure 22 Effect of Ang-(1-7) on PlGF gene expression in triple-negative breast orthotopic tumors 128

Figure 23 Ang-(1-7) reduces VEGF protein expression in a time-dependent manner 130

Figure 24 Ang-(1-7) reduces PlGF protein expression in a time-dependent manner 132

Figure 25 Ang-(1-7) reduces VEGF and PlGF through an

AT(1-7) receptor 134

Figure 26 Ang-(1-7) reduces VEGF and PlGF through an up-regulation in DUSP1 137

Figure 27 Gene knockdown with siRNAs to DUSP1 in MDA-MB-231 cells 139

Figure 28 Ang-(1-7) inhibition of endothelial cell tubule formation 143

Figure 29 Dose-dependent reduction of tubule formation by Ang-(1-7) 145

Figure 30 Ang-(1-7) inhibition of HUVEC endothelial cell

tubule formation 147 Figure 31 Effect of Ang-(1-7) endothelial cell proliferation 150

x

Figure 32 Ang-(1-7) inhibits angiogenesis in vivo. In vivo effects of Ang-(1-7) in the chicken embryo 153

Figure 33 Inhibition of neovascularization in the CAM by Ang-(1-7) is mediated by an AT(1-7) receptor 155

Figure 34 Effect of Ang-(1-7) on human lung cancer xenograft growth 158

Figure 35 Inhibition of human lung tumor angiogenesis

by Ang-(1-7) 163 Figure 36 Reduction of VEGF in human A549 lung

tumor xenografts by Ang-(1-7) 165

Figure 37 The reduction of VEGF by Ang-(1-7) is time-dependent and mediated by an AT(1-7) receptor 167

xi

LIST OF TABLES

CHAPTER I Page

Table I Current Clinical Trials Treating Patients with Triple Negative Breast Cancer 41

CHAPTER II

Table II Preparation of Master Mix for Reverse Transcriptase Reaction. 79

Table III Lowry Protein Assay Standard Curve. 85

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LIST OF ABBREVIATIONS

[D-Ala7]-Ang-(1-7) [D-alanine7]-angiotensin-(1-7)

µg Micrograms

µL Microliter

AC Adenocarcinoma

ACE Angiotensin Converting Enzyme

ACE2 Angiotensin Converting Enzyme 2

AMV Avian Myeloblastosis Virus

Ang I Angiotensin I

Ang II Angiotensin II

Ang-(1-7) Angiotensin-(1-7)

APAF-1 Apoptotic Protease Activating Factor

APS Ammonium persulfate

AT(1-7) Angiotensin type (1-7) receptor

AT1 Angiotensin type I receptor

AT2 Angiotensin type II receptor

ATP Adenosine triphosphate

BAC Bronchioloalveolar carcinoma

BBI Bowman Birk Inhibitor

cAMP Cyclic adenosine monophosphate

CCCWFU Comprehensive Cancer Center of Wake

Forest University

CMV Cytomegalovirus

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CNA Copy Number Alterations

COX Cyclooxygenase

DNA Deoxyribonucleic acid

DOX Doxycycline

DCIS Ductal Carcinoma In Situ

ECM Extracellular Matrix

ECOG Eastern Cooperative Oncology Group

EGF Epidermal Growth Factor

EGFR Epidermal Growth Factor Receptor

ERK Extracellular regulated kinase

FADD Fas associated death domain

FDA Federal Drug Administration

FGF-1 Fibroblast growth factor-1

Flk-1 Fms-like tyrosine kinase-1

GTP Guanosine Triphosphate

H Hour

HER2 Human Epidermal Growth Factor

Receptor

HRE Hypoxia-response element

HuR Hypoxia-induced stability factor

IRES Internal Ribosomal Entry Site

JNK c-Jun amino-terminal kinase

KIM Kinase interaction motif

xiv

Kg Kilogram

LCIS Lobular Carcinoma In Situ

LIF Leukemia Inhibitory Factor

MAP kinase Mitogen-Activated Protein kinase

MMP Matrix Metalloproteinase

MKP Mitogen-activated protein kinase

phosphatase

mRNA Messenger RNA

NEP 24.11 Neprilysin

NF-κß Nuclear factor-κß

NSAIDS Non-Steroidal Anti-Inflammatory Drug

NSCLC Non-Small Cell Lung Cancer

OR Odds Ratio

OS Overall Survival

PAI-1 Plasminogen Activated Inhibitor-1

PARP Poly ADP-Ribose Polymerase

PBS Phosphate-Buffered Saline

pCR pathologic complete reponse

PCR Polymerase Chain Reaction

PDGF Platelet-Derived Growth Factor

PFS Progression Free Survival

PG Prostaglandin

PGES Prostaglandin E Synthase

xv

PGIS Prostaglandin I Synthase

PKC Protein Kinase C

PlGF Placental Growth Factor

POP Prolyl Oligopeptidase

RR Relative Risk

rRNA Ribosomal RNA

RTK Receptor Tyrosine Kinase

SDS Sodium Dodecyl Sulphate

sFlt-1 Soluble Fms-like tyrosine kinase-1

SNPs Single Nucleotide Polymorphism

TEMED Tetramethylethylenediamine

TN Triple Negative

TNBC Triple Negative Breast Cancer

TNT Triple Negative Trial

TOP Thimet Oligopeptidase

TPA Tissue Plasminogen Activator

TSP-1 Thrombospondin-1

USA United States of America

VEGF Vascular Endothelial Growth Factor

VEGFR Vascular Endothelial

Growth Factor Receptor

VSMC Vascular Smooth Muscle Cell

xvi

ABSTRACT

Soto-Pantoja, David Ricardo

ANGIOTENSIN-(1-7): A NOVEL SMALL MOLECULE FOR THE TARGETED TREATMENT OF TRIPLE NEGATIVE BREAST CANCER

Dissertation under the direction of E. Ann Tallant, Ph.D., Professor and Patricia

E. Gallagher Ph.D., Associate Professor

Triple negative breast cancer (TNBC), an aggressive breast tumor that

disproportionately affects young and minority women, is associated with a poor

survival rate regardless of stage at diagnosis due to development of distant

metastases. Tumors from women with TNBC lack estrogen receptors and

progesterone receptors and have basal expression of the human epidermal

growth factor receptor 2 (HER2), severely limiting therapeutic strategies. The

purpose of this study was to determine whether angiotensin-(1-7) [Ang-(1-7)], an

endogenous peptide hormone that activates the AT(1-7) receptor mas, inhibits the

growth of triple negative breast cancer cells and tumors and identify the

molecular mechanisms for the inhibition of cancer cell growth. Injection of MDA-

MB-231 cells into the mammary fat pad of athymic mice resulted in triple

negative breast tumors that were treated for 28 days with either saline or Ang-(1-

7). The average tumor volume from mice treated with the heptapeptide was

approximately 3-fold less than the size of tumors from control animals (170.8 ±

21.4 mm3 vs. 546.7 ± 87.9 mm3; n = 5, p < 0.05) and tumor weight was reduced

from 1.0 ± 0.2 g in the saline-treated mice to 0.5 ± 0.1 g in Ang-(1-7)-treated mice

(n = 5, p < 0.05). The reduced size of Ang-(1-7)-treated tumors was associated

with low immunoreactivity to Ki67, a proliferation marker (84.2 ± 8.2 compared to

xvii

41 ± 7.6), suggesting that Ang-(1-7) attenuates cell growth. Activity of the MAP

kinases ERK1 and ERK2 was reduced in tumors of Ang-(1-7)-treated mice which

was associated with a receptor-mediated increase in the MAP kinase

phosphatase DUSP1 (1.01 ± 0.1 relative gene expression in tumors from saline-

treated mice compared to 1.78 ± 0.13 in tumors from Ang-(1-7)-treated mice).

The decrease in tumor growth of Ang-(1-7)-treated mice was also associated

with a reduction in the endothelial cell marker CD34 (a decrease in vessel

density from 87.8 ± 6.4 in tumors from saline-treated mice to 32.0 ± 7.0 in tumors

from Ang-(1-7)-treated mice, p < 0.05), suggesting that the heptapeptide inhibited

angiogenesis. The reduced vessel density in Ang-(1-7)-treated tumors correlated

with a 59% decrease in placental growth factor (PlGF) and a 72% reduction in

vascular endothelial growth factor (VEGF), effects blocked by pre-treatment with

siRNAs to the MAP kinase phosphatase DUSP1. These results are in

agreement with studies showing that patients receiving subcutaneous

administration of Ang-(1-7) showed a decrease in circulating PlGF. In another

set of studies, Ang-(1-7) reduced the growth of lung tumor xenografts, The Ang-

(1-7)-mediated reduction in lung tumor xenografts was mediated,in part, by a

reduction in tumor angiogenesis and a decrease in VEGF protein and mRNA.

Ang-(1-7) reduced tubule formation by human endothelial cells by more than

40% at a 10 nM dose, a response which was blocked by pre-treatment with the

specific Ang-(1-7) receptor antagonist [D-Pro7]-Ang-(1-7). Moreover, the

heptapeptide significantly reduced new blood vessel formation by more than 50%

in the developing chick chorioallantoic membrane assay, an effect attenuated by

xviii

the Ang-(1-7) receptor antagonist [D-Pro7]-Ang-(1-7). Taken together, these

results suggest that Ang-(1-7) inhibits angiogenesis in vivo through activation of

an AT(1-7) receptor. Based on the anti-proliferative and anti-angiogenic properties

of the heptapeptide, Ang-(1-7) may be an effective, first-in-class compound for

the treatment of triple negative breast tumors targeting the specific AT(1-7)

receptor mas.

1

CHAPTER I INTRODUCTION

The Renin-Angiotensin System:

The renin-angiotensin system (RAS) plays an important role in the control

of blood pressure homeostasis, cardiovascular and renal function, and cell

growth (1;2). The RAS (Figure 1) is a hormone system that is activated when the

enzyme renin is released and cleaves the parent compound angiotensinogen to

the decapeptide angiotensin I (Ang I). The catabolism of Ang I is a point of

divergence in the system, leading to the production of the bioactive peptide

hormones, angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)]. These

peptide products differ in their carboxy termini which leads to counter-regulatory

actions mediated by high affinity binding to distinct membrane-spanning

receptors (3). More recently, a new angiotensin peptide, angiotensin-(1-12), was

identified, which can serve as a precursor of Ang I, Ang II and Ang-(1-7) (4).

Ang II mediates its actions through two different G protein-coupled

receptors, angiotensin type 1 and type 2 receptors (AT1 and AT2, respectively)

(5;6). The formation of the biologically active octapeptide Ang II from Ang I was

widely studied, resulting in the formulation of pharmacological agents, such as

angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers

(ARBs), that modulate its activity and are used to treat cardiovascular-related

diseases. Ang-(1-7) exerts its actions through a G protein-coupled receptor

encoded by the mas gene (7). Many of the physiological actions of Ang II are

counter-regulated by activation of mas by Ang-(1-7) (7). The pharmacological

2

manipulation of the RAS and its biologically active peptide products is a major

achievement in medicine which has led to an increase in life span and quality of

life for patients.

3

Figure 1: The renin-angiotensin system.

4

AT1R

Angiotensin-(1-5)

AAnnggiiootteennssiinnooggeenn

AAnnggiiootteennssiinn II

AAnnggiiootteennssiinn--((11--77))

Renin

AAnnggiiootteennssiinn IIII

ACE

ACEACE2

AT(1-7) R

Nerprilysin; Prolyl Oligopeptidase; Thimet Oligopeptidase

AT2R

5

Pathways for Ang-(1-7) Formation

Ang-(1-7) is a seven amino acid peptide (Figure 2) present in the

circulation and in various tissues at levels comparable to the other active peptide

of the RAS, Ang II (8-10). Angiotensinogen is cleaved by the enzyme renin to

generate Ang I. Ang-(1-7) is formed directly from Ang I by multiple

endopeptidases that cleave the Pro7-Phe8 bond (Figure 3, Panel A) (11). In the

circulation, the metallopeptidase neprilysin, a membrane-bound enzyme located

on the luminal side of the endothelium with access to circulating peptides,

converts Ang I to Ang-(1-7) (12). Ang I is also a precursor of Ang II, generated

by angiotensin converting enzyme (ACE). An increase in the decapeptide Ang I

after inhibition of ACE activity is associated with elevations in Ang-(1-7) in both

animal models as well as in humans (Figure 3 Panel B) (9;13;14).

Ang II is converted to Ang-(1-7) by the carboxypeptidase action of

angiotensin converting enzyme 2 (ACE2) (15). Although ACE2 is a homologue

of ACE, with 40% sequence homology, the enzyme differs from ACE (16). ACE2

is a monocarboxypeptidase while ACE is a carboxydipeptidase. ACE2 activity is

not inhibited by classical ACE inhibitors (17); however, treatment with ACE

inhibitors results in up-regulation of ACE2 and an increase in circulating Ang-(1-

7)(18). Since, Ang-(1-7) is also a substrate for ACE (12), inhibition of ACE

activity also prevents the degradation of Ang-(1-7) to the inactive peptide Ang-(1-

5).

6

Figure 2: Chemical structure of Ang-(1-7).

7

8

Physiological Effects of Ang-(1-7)

Ang-(1-7) produces a set of distinctive physiological responses that often

oppose those of the well-characterized Ang II peptide (8). Ang II increases blood

pressure, is a potent vasoconstrictor, and stimulates thirst and aldosterone

release. The physiological effects of Ang II can be blocked by selective receptor

antagonists for the AT1 receptor or with ACE inhibitors. In contrast, Ang-(1-7)

reduces the blood pressure of hypertensive dogs and rats, alters renal fluid

absorption, causes vasodilation, and participates in the anti-hypertensive

responses to ACE inhibition or AT1 receptor blockade in hypertensive rats (3). In

addition, Ang-(1-7) has direct anti-fibrotic and anti-hypertrophic properties (19).

Experiments carried out by our group as well as others demonstrated that

infusion of Ang-(1-7) is not associated with the side effects of cytotoxic

compounds. Treatment of rodents, such as rats and mice, with Ang-(1-7) does

not change body weight, heart rate or blood pressure (20;21). A 40-fold increase

in the circulating levels of this peptide in rats with heart failure did not produce

any toxic side-effects or anatomic abnormalities (22). Moreover, Ang-(1-7)

toxicity was investigated in human clinical trials studying cytopenia following

myelosuppressive chemotherapy (23). In this study, Ang-(1-7) was administered

daily for 7 days following a period of washout prior the first cycle of

chemotherapy; treatment was re-initiated 2 days after chemotherapy for a period

of 10 days A group of fifteen patients received escalating doses of 2.5, 10, 50,

75 and 100 µg/kg/day of Ang-(1-7) compared to a group of patients who received

filgrastim, a human recombinant granulocyte colony-stimulating factor that

9

enhances the production of neutrophils and is the standard treatment for patients

experiencing cytopenia after chemotherapy. The adverse side effects usually

associated with this treatment, including thrombocytopenia, anemia, and

lymphopenia, were significantly reduced in patients receiving Ang-(1-7) when

compared to the filgrastim-treated group (23). Overall, no dose-limiting toxicity

was observed following Ang-(1-7) treatment. The results of preclinical and

clinical studies suggest that Ang-(1-7) treatment is safe, with limited detrimental-

side effects when administered to patients.

10

Figure 3: Pathways for the formation of Ang-(1-7) and effect of ACE

inhibition.

11

Panel A

Panel B

ACE2

AAnnggiiootteennssiinnooggeenn

Angiotensin I

Renin

ACE

AAnnggiiootteennssiinn IIII AAnnggiiootteennssiinn--((11--77))

Neprilysin, Thimet Oligopeptidase, Prolyl Oligopeptidase

AAnnggiiootteennssiinn--((11--55))

ACE LLeevveellss

ACE2

AAnnggiiootteennssiinnooggeenn AAsspp--AArrgg--VVaall--TTyyrr--IIllee--HHiiss--PPrroo--PPhhee--HHiiss--LLeeuu--VVaall--IIllee--HHiiss--SSeerr--RR

Angiotensin I Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10

Renin

ACE

AAnnggiiootteennssiinn IIII Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8

AAnnggiiootteennssiinn--((11--77)) Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7

Neprilysin, Thimet Oligopeptidase, Prolyl Oligopeptidase

12

The Ang-(1-7) Receptor

The first evidence of a distinct receptor for Ang-(1-7) was based on the opposing

actions of Ang II and Ang-(1-7) (8;24;25). The majority of the responses to Ang-

(1-7) were not blocked by classical AT1 or AT2 receptor antagonists (26;27). [D-

alanine7]-angiotensin-(1-7) ([D-Ala7]-Ang-(1-7)), a modified form of Ang-(1-7) in

which the proline at position seven is replaced by D-alanine, blocked the

responses to Ang-(1-7) but did not oppose pressor or contractile responses to

Ang II (28;29). The reduction in DNA synthesis in vascular smooth muscle cells

(VSMCs) and myocytes by Ang-(1-7) was prevented completely by [D-Ala7]-Ang-

(1-7), indicating that the AT(1-7) receptor mediates the anti-growth response to

Ang-(1-7) (30). Ang-(1-7) also inhibited revascularization in a rodent model of

wound healing; this effect was reversed when Ang-(1-7) treatment was combined

with [D-Ala7]-Ang-(1-7) (31). Treatment of rat VSMCs with Ang-(1-7) prevented

the Ang II-mediated reduction in ACE2 mRNA, which was also blocked by the

selective Ang-(1-7) receptor antagonist [D-Ala7]-Ang-(1-7) (32). Another

selective Ang-(1-7) receptor antagonist, D-Proline7-Ang-(1-7), was generated

which also reverses the pressor and anti-diuretic actions associated with Ang-(1-

7) without affecting Ang II or ACE activity (33).

Santos et al. (34) reported that the orphan G protein-coupled receptor mas

is an Ang-(1-7) receptor. In mas knock-out mice, Ang-(1-7) binding and

functional responses to the heptapeptide were lost. 125I-Ang-(1-7) bound with

high affinity to mas-transfected COS cells and treatment with Ang-(1-7)

13

stimulated arachidonic acid release from CHO and COS cells transfected with

the mas receptor (7). In addition, the ability of Ang-(1-7) to reduce the mitogen-

activated protein (MAP) kinases ERK1 and ERK2 in cultured VSMCs and cardiac

myocytes was blocked by anti-sense oligonucleotides or siRNAs to mas,

demonstrating that mas may be the AT(1-7) receptor mediating the anti-

proliferative response to Ang-(1-7) (35).

Ang-(1-7) treatment caused nitric oxide (NO) production and activated

endothelial NO synthase and Akt in cardiomyocytes (36). The Ang-(1-7)–

dependent NO increase was abolished by pretreatment with [D-Ala7]-Ang-(1-7).

In contrast, Ang-(1-7) did not increase NO in cardiomyocytes isolated from mas-

deficient rats (37); mas ablation was associated with modification of the proteins

which regulate endothelial NO synthase activity, indicating that endothelial NO

synthase and its binding partners are important effectors of the mas-mediated

pathway in cardiomyocytes. Genetic deletion of the mas receptor in FVB/N mice

resulted in dyslipidemia, increased levels of insulin and leptin, and an elevation in

abdominal fat mass (38). Moreover, mas gene-deleted mice were glucose

intolerant and had reduced insulin sensitivity as well as decreased insulin-

stimulated glucose uptake by adipocytes and decreased GLUT4 in adipose

tissue. Mas knockdown also led to increased muscle triglycerides and elevated

expression of cytokines, such as TGF-beta. Angiotensinogen and Ang II

receptor mRNA were higher in mas-ablated animals in comparison with controls

(38). Taken together, these studies demonstrate the critical physiological role of

14

the mas receptor and shed light on the potential role of the Ang-(1-7)/mas axis in

different physiological processes.

Anti-proliferative Effects of Ang-(1-7)

Vascular growth as well as hypertrophy in terminally differentiated cells is

stimulated by Ang II (39;40). In contrast, as observed in Figure 4, Ang-(1-7)

inhibited growth of VSMCs both in vitro and in vivo (1;20;39). In in vitro studies in

VSMCs, Ang-(1-7) caused a dose-dependent reduction in serum-stimulated DNA

synthesis, with a maximum effect at 1 μM (Figure 4). The reduction in DNA

synthesis by Ang-(1-7) was completely blocked by the selective Ang-(1-7)

antagonist, [D-Ala7]-Ang-(1-7), indicating that an AT(1-7) receptor mediated the

anti-growth response to Ang-(1-7) (30). Stimulation of prostacyclin production

and activation of the cAMP-dependent protein kinase were the mechanisms

responsible for the anti-proliferative effect of Ang-(1-7) (41). Ang-(1-7) was

infused into rats with injured carotid arteries, to determine the effect of the

heptapeptide on vascular growth in vivo (Figure 4). Intravenous infusion of Ang-

(1-7) with an implanted mini-pump increased plasma Ang-(1-7) 3-fold over saline

in carotid injured rats and significantly reduced the cross-sectional area of the

neointima (from 0.12 ± 0.02 mm2 to 0.09 ± 0.01 mm2), with no effect on the

uninjured media layer suggesting that Ang-(1-7) only inhibits actively proliferating

cells (20). In addition, Langeveld et al. reported that Ang-(1-7) treatment

significantly reduced neointimal thickness (112 ± 8 versus 141 ± 11 µm; p<0.05),

neointimal area (0.510 ±.05 versus 0.70 ± 0.07 mm2; p<0.05), and percentage

15

stenosis (10.4 ± 1.0 versus 14.0 ± 1.3%; p<0.05) compared with the saline-

treated group following implantation of stents into rat aortas (21).

Ang-(1-7) reduced protein synthesis in myocytes and DNA and protein

production in cardiac fibroblasts, indicating that the heptapeptide regulates

cardiovascular cell growth (30;35). Further, Loot et al. reported that Ang-(1-7)

infusion for 8 weeks in rats following coronary artery ligation improved cardiac

function and was associated with a significant decrease in myocyte size in vivo

(22). Ang-(1-7) caused a significant decrease in the fetal bovine serum- or

endothelin-1-stimulated [3H]-leucine incorporation into cardiac myocytes, an

effect reversed when Ang-(1-7) treatment was combined with its receptor

antagonist [D-Ala7]-Ang-(1-7); Ang II receptor blockers were ineffective in

reversing the Ang-(1-7) actions. Moreover, our laboratory reported that

transfection of cultured myocytes with antisense oligonucleotides to the mas

receptor blocked the Ang-(1-7)-mediated inhibition of serum-stimulated MAP

kinase activation, suggesting that the reduction in cardiomyocyte growth is

through activation of the mas receptor.

Increasing circulating levels of Ang-(1-7) injected through the vena cava in

Sprague-Dawley rats inhibited the in vivo Ang II-mediated activation of ERK1 and

ERK2 in the heart (42); Ang-(1-7) inhibited this pro-growth pathway to

counterbalance the trophic effects of Ang II in the heart. Cardiac fibroblast

growth is also decreased by Ang-(1-7) treatment (43); Ang-(1-7) interacted with

16

specific receptors on adult rat cardiac fibroblasts (ARCFs) to produce potential

anti-fibrotic and anti-trophic effects, reversing the effects of Ang II. Ang-(1-7)

inhibited Ang II stimulated [3H]-proline incorporation in a dose-dependent manner

and pretreatment of ARCFs with Ang-(1-7) inhibited the Ang-II-stimulated

increase in collagen synthesis. In the same study, Ang-(1-7) prevented the

expression of growth factors that are associated with remodeling of the heart,

such as endothelin-1 (ET-1) and leukemia inhibitory factor (LIF), in cardiac

fibroblasts (43).

Ang-(1-7) attenuated cardiac Ang II mediated remodeling in vivo (43).

Sprague-Dawley rats infused with Ang II had increased blood pressure, myocyte

hypertrophy, and myocardial interstitial fibrosis, while co-administration of Ang-

(1-7) resulted in a significant reduction of myocyte hypertrophy and interstitial

fibrosis, without an effect in blood pressure. The anti-remodeling effect of Ang-

(1-7) was partially reversed by co-treatment with [D-Ala7]-Ang-(1-7),

demonstrating that these effects are mediated by an Ang-(1-7) receptor. Thus,

the reduction in DNA synthesis and inhibition of prostaglandin production in vitro

in VSMCs and myocytes as well as inhibition of neointimal growth and

improvement of cardiac function observed in rats after Ang-(1-7) infusion

demonstrates the in vitro and in vivo anti-proliferative effects of this heptapeptide.

17

Figure 4: In vitro and in vivo anti-proliferative effects of Ang-(1-7). In the

left panel, photomicrographs of representative histological cross sections

of rat carotid arteries stained with hematoxylin and eosin 12 days after

injury and treatment with saline or Ang-(1-7): A, Uninjured artery from a

saline-treated rat; B, Injured carotid artery from a rat infused with saline;

and C, Injured carotid artery from a rat treated for 12 days with Ang-(1–7).

Magnification x400. The right panel shows the dose-dependent effect of

Ang-(1-7) and Ang II on the growth of rat aortic VSMC’s (modified from

reference 20).

18

Uninjured, saline, (A)

Injured, saline (B)

Injured, Ang-(1-7) (C)

-10 -9 -8 -7 -60

25

50

75

100

125

50

100

150

200

250

300

350

Ang-(1-7)

Ang II

Peptide Concentration (log M)

Angi

oten

sin-

(1-7

) Gro

wth

Inhi

bitio

n(P

erce

nt o

f Ser

um-S

timul

ated

)

Angiotensin II Grow

th Stimulation

(Percent of Basal)

19

Renin-Angiotensin System and Cancer

Ang II is the most intensively studied peptide of the RAS. Ang II

stimulates cell growth and its receptor is expressed in different cancer

malignancies. The mitogenic effects of Ang II in breast and prostate cancer

involves the stimulation of MAP kinase activities (44). In addition, Ang II

increases the transcription factors c-fos and STAT 3 and activates protein kinase

C in different types of cancer cells (45-47). Ang II also stimulated angiogenesis

in murine models of cancer and increased vascular growth factors in preclinical

models of hepatocellular carcinoma (31;48).

The presence of Ang II in cancer tissues also suggests a role for tissue

RAS activation in tumorigenesis. There is evidence for expression of pro-renin,

ACE, AT1 receptors and AT2 receptors in glioblastoma and abnormal breast

tissue as well as in other malignant tissues (49). Further, a role for angiotensin

peptides in cancer growth was suggested by epidemiological studies. Patients

receiving ACE inhibitors were included as a control group in two studies

assessing the increased risk of cancer in hypertensive patients taking calcium

channel blockers (50;51). In these studies, the relative risk of cancer in ACE

inhibitor-treated patients was 0.73 and 0.79, respectively (50;51).. Moreover, in a

retrospective study of 5207 patients in Scotland, the relative risks of incident and

fatal cancer among 1559 patients treated with ACE inhibitors were significantly

reduced, to 0.72 and 0.65, respectively (52). The relative risk was lowest in

patients with lung, colon, and gender-specific cancers. Additionally, direct use of

20

ACE inhibitors was evaluated in several models of cancer. Preclinical studies

showed that treatment with the ACE inhibitor Captopril caused a significant

reduction in renal cell carcinoma and liver fibrosarcoma (53). Moreover,

treatment with the ACE inhibitor Perlindopril suppressed development of

hepatocellular carcinoma in a murine model (54). These results suggest that

drugs which inhibit ACE may attenuate tumor cell growth (48).

Blockade of angiotensin receptors was also evaluated in tumorigenesis.

Experiments in AT1 receptor knock-out mice showed that tumor growth was

slower in these animals when compared to controls (55;56). Administration of

the angiotensin receptor blocker (ARB) Candesartan was effective in reducing

the growth of engrafted melanoma, fibrosarcoma, metastatic lung tumors,

ovarian cancer cells and prostate cancer cells (47;48;56-58). Also, treatment

with the AT1 receptor blocker Losartan significantly reduced the volume of C6 rat

glioma (59). These studies suggest that pharmacological manipulation of the

RAS may be beneficial in the treatment of certain types of cancer.

Inhibition of Lung Cancer Cell Growth by Ang-(1-7)

Ang-(1-7) significantly reduced the time-dependent growth of human lung

cancer cell lines of the SK-LU-1, A549, and SK-MES-1 (60). Treatment with the

heptapeptide also inhibited DNA synthesis, with EC50’s in the sub-nanomolar

range. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT(1-7)

receptor antagonist [D-Ala7]-Ang-(1-7), while AT1 and AT2 angiotensin receptor

21

antagonists were ineffective. All three lung cancer cell lines contained mRNA

and protein for the AT(1-7) receptor mas, suggesting that mas may also mediate

the anti-proliferative response to Ang-(1-7) in lung cancer cells. Other

angiotensin peptides [Ang I, Ang II, Ang-(2-8), Ang-(3-8) and Ang-(3-7)] did not

reduce mitogen-stimulated DNA synthesis of SK-LU-1 cells, demonstrating the

specificity of the response. The molecular mechanism for the inhibition of lung

cancer cell growth by Ang-(1-7) included a reduction in mitogen-stimulated

ERK1/ERK2 activities, indicating that the anti-proliferative effects may occur, at

least in part, through inhibition of the ERK signal transduction pathway.

Ang-(1-7) was infused into athymic mice injected with human lung cancer

cells in a xenograft model of lung cancer. Following a 28 day infusion of Ang-(1-

7), the size of tumors in mice infused with saline doubled, while the tumors in

mice infused with Ang-(1-7) were significantly reduced as compared to tumor

size prior to treatment (Figure 5). These results correlated with a reduction in the

proliferation marker Ki67 in the Ang-(1-7)-infused tumors when compared to the

saline-infused tumor tissues. Treatment with Ang-(1-7) significantly reduced

COX-2 mRNA and protein in tumors of Ang-(1-7)-infused mice when compared

to mice treated with saline as well as in the parent A549 human lung cancer cells

in tissue culture, suggesting that Ang-(1-7) may decrease COX-2 activity and

pro-inflammatory prostaglandins to inhibit lung tumor growth. There was no

effect of the heptapeptide on COX-1 mRNA in xenograft tumors or A549 cells.

22

Taken together, these results suggest that Ang-(1-7) may serve as an effective

chemotherapeutic agent for lung cancer targeting the mas receptor.

23

Figure 5: Inhibition of human lung tumor xenograft by Ang-(1-7) ( modified

from reference 146).

24

-30 -24 -18 -12 -6 0 6 12 18 24 300

50

100

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200

250

300

350

400

450

Ang-(1-7)Saline

Days

Perc

ent C

hang

e

** *

* **

*

**

Saline Ang-(1-7)

25

Breast cancer

Breast cancer is a leading cause of death among women and is a global

public health concern with more than 1.15 million cases worldwide (61) In the

United States, breast cancer was one of the most commonly diagnosed cancers

in 2007 and will account for more than 30% of new cases, causing the death of

more than 40,000 women (62). Based on the National Cancer Institute

Surveillance Epidemiology and End Results Program (SEER), the incidence of

breast cancer has increased dramatically and is the highest ranking cause of

cancer deaths among women between the ages of 20 to 59 years.

Breast cancer is most prevalent among non-Hispanic white women;

however, lower survival rates were reported for Blacks and Hispanics (63).

Although this may be due to health care access and socioeconomic status, the

rates of incidence and mortality among these ethnic groups may also be linked to

genetic predisposition. Mutations in two autosomal dominant breast cancer

susceptibility genes, BRCA1 and BRCA2, were associated with high risk among

carriers, having a 26% to 84% lifetime risk of developing the disease (64-66).

One percent of the Ashkenazi Jewish population carry mutations in BRCA1 and

BRCA2 that account for 20% and 8%, respectively, of early onset breast cancer

in this population (67), demonstrating the strong correlation of alterations in these

genes and the development of breast cancer. Differences in the prognosis of

breast cancer was attributed to increased mutations on BRCA1 in Caucasian

populations and more frequent mutations on BRCA2 in African-American

26

populations (68). Recently, the Fancomi anemia J gene BRIP1 was identified as

another strong susceptibility gene for breast cancer. It is estimated that BRIP1

mutations confer a 2.0-fold increase in the relative risk of breast cancer (69).

Other risks factors including age, time of menarche, pregnancy, number of breast

biopsies and family history also were associated with the prevalence of breast

cancer. Nonetheless, the principal risk of development of the disease is gender.

The ratio of incidence in male compared to females is 1:100. This is attributed to

ovarian hormonal effects which correlate with studies showing that longer

exposure to estrogen increases the risk of breast cancer (70).

Breast cancer is not a single disease, but a disorder that presents itself

with diverse pathologies and different clinical behaviors. Recognition of the

diversity of breast cancers led to a classification system which categorizes breast

tumors based on the location of the initial tumor, stage, grade and, more recently,

molecular expression profiles. Classification based on location determines

whether the abnormal cells are contained in the ducts of the breast, known as

Ductal Carcinoma in situ (DCIS), or in the lactiferous ducts, known as Lobular

Carcinoma in situ (LCIS). A more rare type of breast cancer is inflammatory

breast cancer in which plaques are present under the breast skin and cause the

blockade of the lymph nodes resulting in inflammation of the breast.

Stages refer to the size of a breast tumor and whether it has invaded

surrounding tissue or distant organs; stages range from 0 to IV. Stage 0

27

describes a tumor that has not invaded surrounding tissue such as DCIS or

LCIS, while stage I depicts a tumor that has invaded surrounding tissue and

measures up to 2 cm. Stage II describes a tumor that has metastasized to the

lymph nodes or a tumor that measures more than 2 cm but has not spread to the

lymph nodes; stage III depicts invasive breast tumors that are larger than 5 cm or

any tumor cell that is found on the lymph nodes near the breast bone, chest wall,

skin, inflammatory breast cancer. Stage IV describes an invasive breast cancer

that has metastasized to distant organs of the body. Tumor grade scores the

tumor from 1 to 3 based on the Bloom-Richardson scoring system in three

different categories, determining the degree of tumor vascularization, mitotic

index and cell morphology.

Molecular profiling of breast tumors, a key advancement in breast cancer

research, identifies tumors by expression of the estrogen receptor (ER), the

progesterone receptor (PR) and/or the human epidermal growth factor receptor

HER2. Molecular profiling also utilizes complementary DNA microarray

technology and hierarchical clustering analysis to identify the unique molecular

signatures of breast tumors. Breast tumors are classified into four basic types,

depending upon these analyses (71-73).

• Normal breast like: The molecular cluster of genes resembles that of

normal epithelial cells of the breast, with a normal adipose tissue pattern,

28

and includes genes such as fatty-acid binding protein 4 and PPARγ

(peroxisome proliferator-activated receptors).

• Luminal A and B types (also known as luminal cell cluster): This type

of epithelial cell is distinguished by immunohistochemistry using

antibodies to keratins 8/18. These tumors usually express the estrogen

receptor and genes that are associated or controlled by estrogen. Luminal

A and B breast tumors are primarily low grade with a low mitotic index.

The gene expression signature is similar to the expression of normal cells

that line the lumen of the ducts and glands of the breast. Luminal A

tumors are ER+ and PR+ and HER2 normal while Luminal B may also

express HER2 as well as a cluster of genes that regulate cell proliferation.

Luminal B tumors also present a higher density of the proliferation marker

Ki67. These tumors respond well to hormonal therapy.

• HER-2 type: These tumors over-express genes found in the ERBB2

amplicon including ERBB2 and GRB7. They tend to be high grade and

have low or no hormone receptor expression. HER-2 tumors respond to

treatment with targeted therapies such as Trastuzumab (Herceptin) and

lapatinib (Tykerb).

• Basal-like: The gene expression profiles of basal-like tumors are

equivalent to those expressed by the cells of the basal layer of the

mammary ducts and glands. These tumors can be distinguished by

expression of markers for cytokeratin 5/6 and the EGFR. Approximately

80 to 90% of these tumors do not express the estrogen receptor or

29

progesterone receptor and have basal expression of HER2 and are often

referred to as triple-negative. However, all basal tumors are not triple

negative. Clinical and prognostic behaviors are currently being

investigated to distinguish triple-negative from other basal like tumors.

Of all the subgroups of breast cancer classified by molecular profiling

analysis, the triple-negative/basal subtype clusters closely indicate uniform

molecular homogeneity at the transcriptional level. Since this dissertation

focuses on the treatment of triple-negative breast cancer, a more detailed

description of the clinical, epidemiological and molecular behaviors is described

in the following section.

30

Figure 6: Molecular profiling of breast tumors (modified from reference 72).

31

Her-2 type

Luminal A

Basal like

Luminal B

Normal Breast

Keratin 5 and 17, Laminin, Fatty acid binding protein 7, P-Cadherin, TRIM29

HER2/c-erb B2 GRB7

ER cluster Proliferation genes

ER alpha, GATA binding protein 3, X-box binding protein 1, Trefoil factor, FOXA1, LIV-1

Adipose tissue enriched pattern

Characteristic Genes

32

Triple-negative Breast Cancer

Breast cancer patients fall into three main groups from a clinical

perspective: those with hormone receptor positive tumors, those with over-

expression of HER2 and those with triple-negative (TN) tumors. The tumors of

patients with triple-negative breast cancer (TNBC) lack estrogen and

progesterone receptors and do not over-express the HER2 gene which severely

limits the treatment options for women diagnosed with this type of breast cancer.

It is estimated that 15% of the anticipated breast cancer cases which will be

diagnosed in 2007 will be of the TN subtype. TNBC mainly impacts pre-

menopausal women, African American women and Hispanic women (74;75). In

a study of 148 Nigerian patients with breast cancer, 66.9% were premenopausal

with a mean age of diagnosis of 43.8, while 78.7% were ER-, and HER2 was

over-expressed in 18.9% of patients, indicating that approximately 59% of the

patients presented either basal-like breast cancer or the TN phenotype. The

Carolina Breast Cancer study, which was designed to recruit more African-

American women, showed that 53% of African-American women had the basal-

phenotype or TNBC (defined in this study as ER–, PR–, HER2 normal,

cytokeratin 5/6 positive, and/or HER1+) compared to 16% in non African-

American women (75). The reasons for the racial differences remain unclear and

may be due to a combination of environmental and genetic factors. A

multivariate analysis that included age, body mass index, hormonal therapy use,

socioeconomic status and ethnicity reported the same prevalence of TN tumors

33

among African-American women, with ethnicity being the highest predictor of

tumor characteristics (72).

An increased number of single nucleotide polymorphisms (SNPs) was

found in basal-like breast cancer when compared to other molecular subtypes

(76). Abnormal expression of SNPs between normal and tumor tissues is

considered a loss of heterozygosity (LOH). Genome-wide SNP arrays

demonstrated that LOH rate is higher in basal-like breast cancer (76). DNA copy

number alterations (CNA’s) serve as prognostic tools that determine genetic

instability in breast cancer. A study using an array-based comparative genomic

hybridization array to profile DNA showed that CNA’s occur at a higher frequency

in basal-like cancers than in other subtypes (77). A commonly found gene region

in ER- and basal-like tumors is the 6p21-p25 which contains several candidate

oncogenes, including DEK, E2F3, NOTCH4, PIM1, and CCND3. In addition, loss

of 5q is found in basal-like breast cancer; DNA repair and tumor suppressor

genes are located in 5q which is commonly lost in BRCA1-like tumors (78). A

BRCA1-modifier locus for hereditary breast cancer penetrance was also mapped

to 5q (77).

An aggressive phenotype is associated with deleterious mutations in

BRCA1. BRCA1 is essential in regulating DNA repair and mutations of this gene

lead to genomic instability and high risk of breast cancer (65) Patients with

BRCA1 mutations have a lifetime risk of developing breast cancer of more than

34

80% (76;79). Tumors that develop in BRCA1 mutations carriers are

characterized by ER negativity and no HER2 expression. Cluster analysis of

microarray RNA expression demonstrated that tumors with familial-BRCA1

mutants strongly segregate with triple-negative sporadic tumors (80). Moreover,

another common characteristic of triple-negative breast tumors is X chromosome

inactivation and X inactive specific protein (XIST) expression which require a

functional BRCA1, a protein defective in both TNBC and BRCA1-associated

breast tumors (79). This suggests that patients with inherited mutations in

BRCA1 often develop breast cancer that is basal-like or of the TN subtype. From

a cohort of 482 patients, 99 were BRCA-tested and 10 showed mutations in

BRCA1; of these 10 patients, 8 were classified as triple-negative (81) Another

abnormality in TN tumors, in addition to aberrations in the BRCA1 pathway, is a

high proliferation rate which is associated with a high index for Ki67, aberrations

in p53, enhancement of MAP kinases activities and dysregulation of cell survival

pathways such as Akt signaling (82-84).

Another critical component of TNBC is the rate of recurrence (76). TNBC

often re-occurs within the 1st and 3rd years following diagnosis with most deaths

occurring 5 years following therapy. This subtype of breast cancer is also

characterized by its ability to form distant metastasis (79-81). The majority of

triple-negative breast cancers are high-grade invasive ductal carcinomas and

patients have significantly shorter survival following the first metastatic event

when compared to other subgroups of this malignant disease.

35

Clinical Characteristics of Triple-Negative Breast Cancer

Triple-negative (TN) tumors are characterized by high nuclear grade (based on

the Bloom-Richardson Scaling System), an increased incidence of metastasis

and shorter recurrence-free survival when compared to other subtypes of breast

cancer (75). The Carolina Breast Cancer Study compared basal-like breast

tumors to luminal type A tumors and concluded that basal-like tumors had more

p53 gene mutations (44% vs. 15%, P<.001), a higher mitotic index (odds ratio

[OR], 11.0; 95% confidence interval [CI], 5.6-21.7), more marked nuclear

pleomorphisms (OR, 9.7; 95% CI, 5.3-18.0), and higher combined grades (OR,

8.3; 95% CI, 4.4-15.6) (75). Morphologically, TN tumors are characterized by

pushing borders of invasion, geographic tumor necrosis, central sclerosis,

lymphocytic response, spindled tumor cells, squamous metaplasia, high nuclear

grade, high mitotic count, high nuclear to cytoplasmic ratio and metaplastic

features (79). The clinical management of TNBC is difficult since these tumors

do not respond to classical hormonal therapy regimens. The prognosis of TN

breast cancer patients is lower compared to the other subtypes, due, in part, to

the lack of response to targeted therapy and the characteristic aggressiveness of

these tumors (84). However, TN tumors do respond better to chemotherapy than

the other subgroups. A study examining the response of patients with TNBC to

neoadjuvant chemotherapy demonstrated that pathologic complete response

(pCR) rate is higher in individuals with TNBC than non-TNBC patients (74). On

the other hand, TNBC patients with residual disease after neoadjuvant

36

chemotherapy had significantly shorter post-recurrence survival than patients

with non-TN tumors and residual cancer (74).

Clinical studies also showed that the risk of recurrence is time-dependent.

The risk of relapse, metastasis and death is higher for TNBC patients during the

first three years after therapy, when compared to non-TNBC subjects; the risk for

relapse after three years for TNBC patients is actually lower than non-TNBC

suggesting that the highest risk for relapse in this group is 4 to 6 years after

treatment (74;80). Relapse in TN patients has a worse prognosis when

compared to other breast cancer subtypes. In addition, there is a high rate of

brain and lung metastasis in patients with TNBC, with a noticeable increase in

brain metastasis from tumors that express EGFR and basal cytokeratins (72).

On the other hand, basal phenotype tumors showed a lower rate of lymph node

metastasis when compared to other molecular subtypes of cancer (79).

Treatment of Triple-negative Breast Cancer

Even though there are several therapies under clinical investigation (Table

I), the only course of treatment currently available for patients with TNBC is

chemotherapy. Retrospective studies demonstrated that TNBC patients have

twice the benefit of cytotoxic chemotherapy compared to patients with ER+

breast cancer (72). A complete pCR rate to preoperative Placlitaxel and

doxorubicin was seen in 45% of basal-like cancer, and only 6% of luminal

cancers; this highlights the high chemosensitivity to cytotoxic therapy observed in

37

triple-negative breast cancer patients. Basal-like cancers and TNBC show high

rates of response to neoadjuvant anthracycline and taxane neoadjuvant

chemotherapy(85) which contradicts studies showing that tumors expressing

mutated p53, such as TNBC, are resistant to taxanes.

Most of the tumors that arise from BRCA1 mutations are TN, suggesting

that a common method of treatment could be designed for patients with TNBC or

BRCA1 mutations. BRCA1 defective tumors are sensitive to cross-linking agents

such as platinum salts (86). The Triple-Negative Trial (TNT) compares the

platinum agent carboplatin to docetaxel in women with advanced sporadic TNBC

and women with defined genetic malfunctions in BRCA1 and BRCA2 to

determine if TNBC patients are sensitive to docetaxel (85). BRCA1 dysfunction

can be directly targeted with poly ADP-ribose polymerase inhibitors (PARP) (76).

Single-stranded breaks are usually repaired by the base excision repair pathway

of which PARP1 is one of the main components. In the absence of this pathway,

single stranded breaks degenerate to double-strand breaks, which are not

repaired in BRCA1 null cells. In vitro data showed that PARP1 inhibition results

in highly selective apoptosis of BRCA1 null cells and these inhibitors are

currently under study in phase I clinical trials (87;88).

Trastuzumab, a monoclonal antibody against HER2, is effective in the

treatment of breast cancer patients that over-express HER2. In addition, there

are other genes that are expressed in TNBC which may be targeted for the

38

treatment of TNBC, such as the EGFR. Greater than 66% of patients with basal

phenotype tumors and TNBC express EGFR (85). Ongoing studies are

addressing the response of current targeted therapies to EGFR such as

Cetuximab, alone or in combination with carboplatin, or drugs which target EGFR

receptor phosphorylation such as Gefitinib or Erlotinib, in patients with TN

subtype. A phase III open label clinical trial from the Eastern Cooperative

Oncology Group (ECOG 2100) investigated the efficacy of Placlitaxel alone or

Placlitaxel in combination with Bevacizumab in breast cancer patients; the TN

subset greatly benefited from the combination compared to other subtypes of

breast cancer, suggesting a possible combinatorial treatment for TNBC (72;89).

The biology of TN tumors is heterogeneous and complex and creates challenges

in its clinical management. Even though TNBC patients respond well to

neoadjuvant chemotherapy, the Overall Survival (OS) is still very poor and calls

for the formulation of novel chemotherapeutic agents to target this aggressive

subtype of breast cancer.

Chemoprevention of Triple-negative Breast Cancer

The identification of breast cancer risk factors has led to the investigation of

preventive approaches aimed at decreasing disease morbidity and mortality.

Risk assessment models were developed to determine the likelihood of

individuals developing breast cancer and provide advice on options to prevent

the disease. In general, individuals at high risk for development of breast cancer,

including TNBC, can take preventive measures that include prophylactic surgery

39

of the breasts and ovaries. While these procedures significantly reduce risk of

breast cancer, the results are permanent and do not guarantee that cancer will

not develop. Randomized clinical trials, using SERMs such as tamoxifen,

showed the value of breast cancer chemoprevention in patients with hormone-

responsive tumors. Tamoxifen is the first chemopreventive drug for women at

high risk approved by the Food and Drug Administration (52). However, no

significant chemoprotection was observed for women with BRCA 1 gene

polymorphisms and women who are not sensitive to hormonal therapy (78).

Other chemopreventive drugs, such as aromatase inhibitors, were developed to

modify circulating estrogen levels. Aromatase inhibitors are well-tolerated but

are only prescribed to postmenopausal women with early-stage breast cancer

because of treatment-related amenorrhea (90).

Patients who do not respond to current targeted pharmacological

strategies can greatly benefit from changes in their lifestyles. Evidence from the

Women’s Health Study in Boston showed that women who exercise regularly

have a lower risk of recurrence than women who do not exercise regularly (91).

A study designed to determine whether lowering fat intake would benefit the

outcome for early stage breast cancer patients showed that dietary modification

had a significant impact on relapse-free survival in women with ER-negative

cancer (HR = 0.58; 95% CI = 0.37 to 0.91) compared to women with ER-positive

disease (HR = 0.85; 95% CI = 0.63 to 1.14; interaction test, P = .15 (91). Even

though high risk patients for triple-negative breast cancer can benefit from

40

lifestyle changes to prevent recurrence this fraction of breast cancer patients still

has the poorest relapse free survival. These studies indicates the urgent need to

elucidate genetic markers expressed by TN tumors with the objective of

formulating novel pharmacological chemopreventive strategies that aim to lower

the high risk of recurrence and relapse in TNBC patients.

41

Table I:

Current Clinical Trials Treating Patients with Triple Negative Breast Cancer

Sponsor and Study ID

Intervention Objective

Astra Zeneca NCT00707707

AZD2281 Determine efficacy of AZD2281 in combination with paclitaxel.

Bristol-Myers Squibb NCT00371254

Dasatinib (BMS-354825)

Determine effectiveness of Dasatininb in treatment of women with progressive TNBC.

BEATRICE Hoffmann-La Roche

NCT00528567

Bevacizumab and Standard adjuvant

chemotherapy

Evaluate the efficacy and safety of the addition of bevacizumab to standard adjuvant therapy in patients with TNBC .

Leo W. Jenkins Cancer Center NCT00542191

Doxorubicin Cyclophosphamide

Paclitaxel Carboplatin

Measure response (DFS and OS) of patients with primary and axillary lymph nodal TN tumors to neoadjuvant chemotherapy.

University of Kansas Genentech

NCT00491816

Erlotinib To assess the pathological pCR with 4-6 cycles of neoadjuvant chemotherapy plus erlotinib in patients with TNBC.

Triple Negative Breast Cancer Trial

(TNT) NCT00532727

Carboplatin Docetaxel

To determine whether there is greater activity for carboplatin than a taxane standard of care (docetaxel) in women with TNBC.

Duke University Genentech

NCT00479674

Abraxane Bevacizumab Carboplatin

Determine whether combination of weekly Abraxane® and carboplatin plus biweekly bevacizumab will lengthen time to progression without producing intolerable toxicity.

Massachusetts General Hospital NCT00483223

Cisplatin Determine effectiveness of cisplatin in slowing progression of TNBC and evaluate biomarker to determine which breast cancers breast cancrespond to cisplatin chemotherapy.

Emory University NCT00674206

Gemcitabine and oxaliplatin

Assess if the combination of gemcitabine and oxaliplatin is effective for TNBC.

BiPar Sciences NCT00540358

Gemcitabine, gemcitabine,

carboplatin + BSI-201

This study will determine whether inhibiting PARP-1 activity with BSI-201 in combination with standard chemotherapy is effective for TNBC.

M.D. Anderson Cancer Center NCT00499603

Paclitaxel: 5-Fluorouracil

Epirubicin Cyclophosphamide,

RAD001

Determine if the addition a mTOR inhibitor to standard neoadjuvant chemotherapy in patients with TNBC causes molecular changes of the PI3K/PTEN/AKT pathway.

Bristol-Myers Squibb NCT00633464

Ixabepilone, ixabepilone+ cetuximab

The purpose of this study is to estimate the response rate of ixabepilone monotherapy, and the combination if ixabepilone plus cetuximab as first-line treatment if subjects with TN locally advanced non-resectable and/or metastatic breast cancer.

M.D. Anderson Cancer Center NCT00739063

Tarceva Assess the clinical efficacy, biologic effects and safety of the EGFR inhibitor erlotinib in the treatment of patients with EGFR-overexpressing 'triple receptor-negative' metastatic carcinoma of the breast.

MediGene NCT00448305

EndoTAG-1 + paclitaxel

EndoTAG-1 Paclitaxel

Assess the efficacy, safety and tolerability of a therapy with EndoTAG-1 + paclitaxel in combination and EndoTAG-1 alone as a rescue therapy for patients with relapsed or metastatic triple receptor negative breast cancer.

Pfizer NCT00246571

SU011248 Chemotherapy

The purpose of this study is to compare progression free survival for SU011248 versus standard of care therapy in patients with previously treated, advanced, TNBC locally recurrent or metastatic breast cancer.

M.D. Anderson Cancer Center

Bristol-Myers Squibb NCT00420329

Carboplatin Cetuximab

Determine OR rate to single agent cetuximab in TN metastatic breast cancer and determine OR rate to combination cetuximab plus carboplatin in TNBC.

Source: U.S National Institute of Health, clinicaltrials.org

42

Angiogenesis

Angiogenesis is a complex physiological event in which new blood vessels

develop from the pre-existing vasculature. Angiogenesis should not be confused

with vasculogenesis in which new vessels are formed de novo from endothelial

stem cells or endothelial cell precursors that together form the foundation of a

vessel (92). Angiogenesis is active during development but is generally

quiescent in the normal adult, except during the female reproductive cycle or

following tissue injury (93). Angiogenesis is a tightly controlled process that

requires regulation of the balance between angiogenic-stimulatory and anti-

angiogenic signals which, under normal physiological circumstances, results in

the stability of the mature vasculature (94). However, the balance between pro-

angiogenic and anti-angiogenic factors is disrupted under certain pathological

conditions including inflammatory disorders, wound healing, and tumors.

Tumors require the formation of new blood vessels from preexisting ones

in order to develop, a concept initially proposed by Dr. Judah Folkman in the New

England Journal of Medicine in 1971 (95). Dr. Folkman’s work as well as studies

by other investigators showed that local uncontrolled release of angiogenic

growth factors coupled with dysregulation of endogenous angiogenic inhibitors

results in alterations in the angiogenic balance which are responsible for the

endothelial cell growth that takes place during tumor neovascularization and

other angiogenesis-dependent diseases (94;96). The process of tumor

angiogenesis (Figure 7) is complex, requiring the simultaneous interaction of

43

angiogenic inducing agents, multiple types of cells, endothelial cell receptors,

and elements of the extracellular matrix which result in neovascularization and

enhancement of tumor growth (92;97).

Tumor angiogenesis is characterized by the formation of a tortuous

abnormal vasculature with altered interactions between endothelial cells and

perivascular cells such as pericytes, irregular blood flow, increased vascular

permeability, and incomplete vessel development (98). The “angiogenic switch”

or the disruption of the balance between pro- and anti- angiogenic factors is

triggered by physiological stimuli, such as hypoxia, which is a result of the

increase in cell growth driven by oncogene activation or tumor-suppressor

mutations (99). Initially, tumors grow and reach a critical mass, defined as a

threshold of 0.2 mm, in which oxygen can no longer effectively circulate to all the

cells within the tumor (92). This creates a hypoxic environment which causes

cells within the tumor to produce factors that bind to receptors on endothelial

cells, stimulating the detachment of perivascular cells and dilating the pre-

existing vessel; activated endothelial cells subsequently release proteases which

degrade the basement membrane, allowing endothelial cell migration and

proliferation and resulting in a vessel sprout. Vessel maturation occurs with the

recruitment of perivascular cells. The new vessel nourishes hypoxic and necrotic

areas of the tumor, increasing tumor growth and mass. At the molecular level,

the angiogenic switch is driven primarily by oncogenic activation that results in

the release of pro-angiogenic proteins, such as VEGF, basic fibroblast growth

44

factor (bFGF), interleukins, and transforming growth-factor-β (TGF-β) (100;101).

The tumor microenvironment is hypoxic and the reduced oxygen tension

activates hypoxia inducible factor-1α (HIF-1α), which promotes the transcription

of additional angiogenic factors (102;103).

Regardless of the mechanism which initiates the angiogenic process, pro-

angiogenic growth factors are released from the tumor cells and bind to receptors

on the endothelial cells, causing their activation. This results in up-regulation of

matrix metalloproteinase (MMPs) that degrade the basement membrane and

stimulate proliferation of endothelial cells (104). Cells migrate by chemotaxis,

causing endothelial cell remodeling and tube formation from the pre-existing

vasculature surrounding the tumor, transforming the tumor from a benign to an

invasive state (92).

45

Figure 7: Tumor angiogenesis.

46

Endothelial cell activation

Production and release ofangiogenic factors i.e bFGF, VEGF, PlGF

Endothelial cellproliferationand migration

BLOOD VESSEL GROWTH-sprouting-tube formation

TUMOR GROWTH

Metastasis

Growth factors Cancer cell

Endothelial cell

Tumor Angiogenesis

Endothelial cell receptor

Endothelial cell activation

Production and release ofangiogenic factors i.e bFGF, VEGF, PlGF

Endothelial cellproliferationand migration

BLOOD VESSEL GROWTH-sprouting-tube formation

TUMOR GROWTH

Metastasis

Growth factors Cancer cell

Endothelial cell

Tumor Angiogenesis

Endothelial cell receptor

47

Vascular Endothelial Growth Factor

The family of vascular endothelial growth factors (VEGF) is a group of

cytokines that are involved in essential physiological processes and are

aberrantly expressed in many pathologies. The original member of this family is

VEGF-A which was initially discovered as a vascular permeability factor and is

widely expressed in tumors (105). Other isoforms include VEGF-B which is

primarily found in the heart, VEGF-C and D which are essential in the regulation

of lymphangiogenesis, VEGF-E which is a viral protein that behaves like VEGF-A

and Placental Growth Factor (PlGF), originally discovered in the placenta and

important during embryonic development as well as in other pathologies

including cancer (discussed below) (98;106). In addition, alternate sites of

splicing of VEGF mRNA in exons 6, 7 and 8, results in the formation of alternate

forms of VEGF with different binding affinities and amino acid numbers, including

VEGF121, VEGF121b, VEGF145, VEGF165, VEGF165b, VEGF189, VEGF206. These

variants have different functions which determines whether they are pro-

angiogenic (variants arising from the proximal splice site), expressed during

angiogenesis, or anti-angiogenic (variants which come from distal splice site).

For example, VEGF165b is anti-angiogenic, endogenously inhibits VEGF-A and is

down-regulated in certain tumors (98). Moreover, the splicing at exons 6 and 7

dictates the interaction of VEGF with heparan sulfate proteoglycans (HSPGs)

and neuropilin co-receptors, allowing the binding and activation of VEGF

receptors.

48

Members of the VEGF family of cytokines bind with high affinity to three

receptor tyrosine-kinases, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-

3 (FLT-4). VEGFR2 is normally expressed in higher copy number than VEGFR;

however, both receptors are over-expressed in pathological angiogenesis (98).

Plasma forms of VEGF, present in the circulation, as well as soluble truncated

forms of both VEGFR1 and VEGFR2, were also identified (98). One of the most

well-characterized soluble forms of VEGF receptor is sFlt-1 (sVEGFR1). sFlt-1 is

produced by alternative splicing and retains VEGF-A and PlGF binding affinity

(98). sFlt-1 in the serum of preeclamptic women prevented endothelial cells

isolated from the human umbilical vein from forming tubes in vitro, demonstrating

the negative regulation of angiogenesis by this form of the receptor (107). VEGF

also binds to two non-tyrosine kinase receptors known as neuropillin-1 and 2.

These receptors mediate the signaling of semaphorins and collapsin and are

involved in development of neuronal systems; neuropillin-1 and 2 are also found

in normal and tumor tissue (108).

Wound healing, inflammatory disorders and tumors also stimulate

neovascularization by a process known as pathological angiogenesis. In the

laboratory setting, pathological angiogenesis was stimulated by the single

activation of VEGF-A (98), demonstrating the powerful effects that VEGF elicits

on aberrant angiogenesis and the importance of this cytokine as a target for

tumor growth.

49

The effects of VEGF are diverse and are time-dependent. VEGF elicits

receptor phosphorylation and stimulates downstream signaling cascades,

vascular dilation, vascular permeability, extravasation and clotting signaling, in a

matter of seconds to minutes (109). In terms of hours and days, VEGF can

reprogram gene expression to stimulate angiogenesis, arteriogenesis, activation

of endothelial cell survival, migration and tubule formation and is also implicated

in the mobilization of endothelial progenitor cells (97). VEGF expression is

regulated by an increase in hypoxia or a reduction in pH; hyperoxia down-

regulates VEGF at both transcription and translation (93). VEGF expression is

also stimulated by hormones, such as estrogen, progesterone and thyroid

hormones. Other cytokines, including members of the TGF family, EGF, PDGF,

and prostaglandins, induce transcription of VEGF (98). Oncogenes and tumor

suppressor genes such as VHL and p53 are especially involved in the activation

of VEGF with or without the requirement of low pH and/or hypoxic conditions

(103). VEGF activation not only stimulates pro-angiogenic pathways but also

leads to stimulation of cell survival pathways, such as the Akt pathway, and can

activate MAP kinase activities which in turn stimulates gene transcription that

activates cell proliferation (97).

Based on the diverse actions of VEGF in angiogenesis and cell survival,

its expression is associated with poor prognosis in many cancers. Therefore,

anti-VEGF therapy has emerged in recent years as a viable option for many

50

cancer patients. Bevacizumab (Avastin) is a monoclonal antibody that targets

VEGF and is the first FDA approved anti-angiogenic agent (110). However,

Bevacizumab alone exhibited subtle anti-tumor activity and studies show the

drug is more beneficial if used in combination chemotherapy (111).

Placental Growth Factor

PlGF is a member of the VEGF family of cytokines originally discovered in

the placenta. PlGF contains 149 amino acids and has 53% homology with the

PDGF region of human VEGF (98). Like VEGF family members, PlGF is

involved in the regulation of blood vessel formation and permeability mediated by

two tyrosine kinase receptors. There are several forms of PlGF including PlGF-

1, PlGF-2 and PlGF-3. PlGF is involved in the control and stimulation of

angiogenesis (93). Carmeliet et al. demonstrated the effect of PlGF on

angiogenesis by deleting exons 3-6 of this gene in mice; their studies

demonstrated that embryonic angiogenesis was not affected by deficiency of

PlGF (112), The role of PlGF in normal adults was restricted to pathological

angiogenesis since the loss of PlGF impairs angiogenesis, plasma extravasation

and collateral growth during ischemia, inflammation, wound healing and cancer.

PlGF may enhance pathological angiogenesis by activating intracellular

signal transduction through activation of the VEGFR-1 and increasing the levels

of VEGF-A binding to VEGFR2 by displacing VEGF-A from VEGFR-1 (113). In

addition, stimulation of VEGFR-1 by PlGF results in intermolecular

51

transphosphorylation of VEGFR-2 that could increase VEGF-A mediated

angiogenesis. Another proposed mechanism is the formation of PlGF/VEGF-A

heterodimers which are elevated in tumors and stimulate angiogenesis through

VEGFR-2 during hypoxic conditions.

Increased expression of PlGF in tumors leads to directs effects on

endothelial cell growth, causing the formation of a tortuous vasculature by

induction of intracellular responses to PlGF as well as by amplifying VEGF-driven

angiogenesis (93) Moreover, PlGF over-expression results in the development

of significant angiogenesis in a diverse number of tissues (114). PlGF also

functions as a chemoatractant for inflammatory cells, such as macrophages and

also bone-marrow-derived cells, a key hallmark of angiogenesis during

pathological conditions such as cancer (115-117). Beyond its role in

angiogenesis, PlGF is implicated in metastatsis; addition of PlGF stimulates in

vitro motility of human breast tumor cell lines (114). In a glioma tetracycline-

regulated PlGF mouse model, over-expression of this growth factor caused an

increase in survivin, resulting in the inhibition of apoptosis and increase in cell

survival (118). Since adult PlGF expression occurs only during pathological

circumstances, inhibition of this growth factor may be beneficial in cancer

treatment.

52

Angiogenesis and Breast Cancer

Breast cancer cells disrupt the balance of angiogenic growth factors and

inhibitors to initiate the angiogenesis process in early carcinogenesis (100).

Primary breast tumor progression and development are dependent on the

formation of new blood vessels from the pre-existing vasculature (100;119). The

aggressiveness and malignancy of breast tumors strongly correlates with

intratumoral vessel density, such that ductal carcinomas in situ were renamed

intraepithelial neoplasia to emphasize their non-life-threatening nature (120).

Microvessel density, specifically increased intratumoral vascularization, is

associated with poor disease-free survival of breast cancer patients (100). The

angiogenic switch occurs early in breast cancer and over-expression of pro-

angiogenic molecules correlates with a poor prognosis of this disease (121). For

example, breast tumors over-expressing the Her-2/Neu oncogene are invasive

and up-regulate the production of pro-angiogenic growth factors, such as TGF-β,

angiopoietin-1 and plasminogen-activator inhibitor-1 (122). Moreover,

intratumoral microvessels of breast tumors over-express a number of pro-

angiogenic factors, including MMP-2 and -9, E-cadherin, integrins and pro-

angiogenic receptors, such as Flk-1 and KDR which bind VEGF (100;100). The

hypoxic environment produced by the breast cancer cell increases the

expression of VEGF and its isoforms. The addition of estradiol causes the

accumulation of VEGF mRNA and protein in cultured medium of estrogen-

dependent cell lines(123). In addition, tissue VEGF expression correlates with

the degree of tumor vascularity in primary breast cancer(89).

53

Expression of endogenous inhibitors of angiogenesis plays an important

role in the growth of breast cancer cells. Transfection of thrombospondin-1 into

breast cancer cells causes a reduction in tumor growth and metastasis (124).

Increased aggressiveness of a primary breast tumor involves a disruption in the

balance of MMPs and tissue inhibitors of MMPs (TIMPs) causing proteolytic

degradation of the basement membrane (125). A correlation of the ratio of MMP-

2 and TIMP-2 and the nodal status of the breast tumor suggests that an

alteration in the balance of MMPs and TIMPs facilitates the spread of tumor cells

into the circulatory system and lymphatic ducts (126).

The use of anti-angiogenic drugs such as Bevacizumab (Avastin™) was

investigated in the treatment of breast cancer. Bevacizumab is a monoclonal

antibody directed against VEGF, a potent stimulator of angiogenesis (127).

Currently, Bevacizumab is FDA-approved for the treatment of colon cancer (110)

and the drug was investigated for the treatment of renal, lung (128) and other

non-oncologic malignancies (129). In breast cancer, the use of Bevacizumab

showed promise in phase II clinical trials of refractory breast cancer and in phase

III clinical trials when used in combination with chemotherapy (128).

Unfortunately, like many other therapies, administration of Bevacizumab was

associated with detrimental effects that include gastrointestinal perforations,

hemorrhage, wound healing difficulties, hypertension and cardiovascular

complications, limiting its usefulness (129).

54

Role of Angiotensin-(1-7) in Angiogenesis

Ang-(1-7) or agents that increase Ang-(1-7) in the circulation inhibit

angiogenesis (53;130). Captopril, an anti-hypertensive drug that increases

circulating Ang-(1-7),(14) inhibited bFGF-induced neovascularization in the rat

cornea (130). Captopril also acted directly on capillary endothelial cells to inhibit

migration by blocking their chemotaxis towards an inducer of angiogenesis (130).

Captopril caused anti-tumor activity in rats injected subcutaneously with

methylcholanthrene-induced sarcoma cells (53). Direct inhibitory effects of Ang-

(1-7) on angiogenesis were observed in a murine sponge model of angiogenesis,

an in vivo technique representative of the formation of new blood vessels from

pre-existing ones(25;31). In this model, a cannulated sponge disk was implanted

subcutaneously in the dorsa of mice to induce a wound repair response. Ang-(1-

7) infusion reduced hemoglobin content, blood flow and proliferative activity in

the disc containing vehicle. The effect of Ang-(1-7) on the formation of new

blood vessels was inhibited by the Ang-(1-7) receptor blocker [D-Ala7]-Ang-(1-7).

Furthermore, the anti-angiogenic response was blocked by pre-incubation with

either aminoguanidine or NG-nitro-L-arginine methyl ester (L-Name), indicating

that the observed anti-angiogenic response is mediated by nitric oxide (NO) (31).

Ang-(1-7) stimulates the release of NO from bovine aortic endothelial cells and

causes the vasodilation of blood vessels through an endothelial release of NO

(2). While this is a model of angiogenesis during wound healing, it suggests that

55

Ang-(1-7) may inhibit the neovascularization that supplies nutrients for the growth

and expansion of breast tumors.

Mitogen Activated Protein Kinases

The essential role of Mitogen Activated Protein Kinases (MAP Kinases) in

eukaryotic signal transduction as well as a wide variety of biological processes is

well established. These protein kinases respond to extracellular stimuli by

regulating gene expression, cell growth and survival (Figure 8) and are

deactivated by MAP kinase phosphatases (131). There are at least four distinctly

regulated groups of MAP kinases: extracellular signal-related kinases (ERK)-1/2,

Jun amino-terminal kinases (JNK1/2/3), p38 proteins (p38 / / / ) and ERK5

(132). These MAP kinases are activated by specific MAP kinase kinases

(MAPKKs): MEK1/2 for ERK1/2, MKK3/6 for the p38, MKK4/7 (JNKK1/2) for the

JNKs, and MEK5 for ERK5 (Figure 7). However, the signaling cascade is more

complex since each MAPKK can be activated by more than one MAP kinase

kinase (MAPKKK), conferring responsiveness to distinct stimuli (131). For

example, activation of ERK1/2 by growth factors depends on the MAPKKK c-Raf,

but other MAPKKKs activate ERK1/2 in response to pro-inflammatory stimuli

(131).

A cascade of kinases is triggered downstream of Ras, including the

activation of cell surface receptors by growth factors that elicit numerous cellular

responses which stimulate cell proliferation and cell differentiation (131;132).

56

Ras couples with GTP in its effector loop domain. Amino acid substitutions in

this effector loop create three alternative forms of the Ras protein which interact

with three different downstream effectors--PI3K, Raf or Ral-GEF. Raf activates

proteins by protein phosphorylation of serine and threonine residues. The

association of Raf and Ras causes a change in the three dimensional

configuration of the Raf kinase molecule which allows the phosphorylation and

activation of MEK (MAPKK). MEK phosphorylates serine/threonine residues as

well as tyrosine residues. The dual specificity kinase activity of MEK activates

and phosphorylates ERK1 and ERK2 in the common –TXY motif. ERK1 and

ERK2 are functionally redundant, but some studies demonstrate a difference in

their substrate specificity (133;134)

Active ERK kinases phosphorylate cytoplasmic substrates and can also

translocate to the nucleus where they elicit phosphorylation of transcription

factors. MAP kinases regulate cell proliferation; ERK1/2 stimulates DNA

synthesis through phosphorylation of carbamoyl phosphate synthetase II, a rate-

limiting enzyme in pyrimidine nucleotide biosynthesis. In addition, the ERKs,

probably through MAPKAPKs (Mitogen-Activated Protein Kinase-Activated

Protein Kinase) such as RSK (ribosomal s6 kinase), promote cell-cycle

progression by inactivating MYT1, a cell-cycle inhibitory kinase, but arrest meiotic

cells at metaphase II by activating a cytostatic factor. The ERKs can also

stimulate cell proliferation indirectly by enhancing AP-1 activity, resulting in cyclin

D1 induction. The ERK MAP kinase pathway alone is insufficient for initiation of

57

DNA synthesis and requires assistance from phosphatidylinositol-3-OH kinase,

which is indirectly activated by autocrine growth factors whose expression is ERK

responsive. Autocrine factors are important in cellular responses to MAP kinases

and provide a means for one MAP kinase cascade to activate other signaling

pathways. In T cells, the JNK pathway may stimulate proliferation by inducing IL-

2 expression (131). The mechanism by which ERK protects cells from apoptosis

is complex. In cerebellar granular cells, ERK activation by survival factors

prevents apoptosis through RSK, which inactivates the pro-apoptotic protein BAD

(135;136).

ERK may also induce growth factors that promote cell survival. The

completion of this cascade of phosphorylation events significantly contributes to

cell phenotypes that are effectors of the Ras oncoprotein. In cancer, the proteins

downstream of Ras are highly activated or dysregulated which leads to aberrant

cell proliferation, inhibition of cell death, and loss of contact inhibition. Therefore,

the inactivation of this pathway either by inhibition of factors that couple to

receptors or the downstream protein kinases is an active area of research that

holds promise for the treatment of many types of cancer.

58

Figure 8: Mitogen-activated protein kinase pathways.

59

RafMEKK 1-4, MLK3

MEK 1,2

ERK 1,2

MAPKAP p90RSK

ELKCREB

MEKK 2,3

MEK 5

BMK1/ERK5

SGK BAD

CX43Sap1a

ASK1 TAK1MEKK, MLK3

MEKK 3,6

p38 α,β,γ,δ

MEF2 PRAK

ATF2

MLK2 DLK2MEKK 1-4

MEKK 4,7

JNK 1,3

C-JUN

Cellular Responses: Proliferation, Differentiation, Survival, Apoptosis

Mitogens(GFs, Cytokines)

Stresses(Oxidation,UV)

Cell Membrane

RafMEKK 1-4, MLK3

MEK 1,2

ERK 1,2

MAPKAP p90RSK

ELKCREB

MEKK 2,3

MEK 5

BMK1/ERK5

SGK BAD

CX43

BAD

CX43Sap1a

ASK1 TAK1MEKK, MLK3

MEKK 3,6

p38 α,β,γ,δ

MEF2 PRAK

ATF2

MLK2 DLK2MEKK 1-4

MEKK 4,7

JNK 1,3

C-JUN

Cellular Responses: Proliferation, Differentiation, Survival, Apoptosis

Mitogens(GFs, Cytokines)

Stresses(Oxidation,UV)

Stresses(Oxidation,UV)

Cell Membrane

60

Mitogen-Activated Protein kinase Phosphatases

MAP kinases phosphatases belong to a group of dual-specificity protein

phosphatases that can dephosphorylate both phospho-threonine and phospho-

tyrosine residues, negatively regulating MAP kinase signaling (137). This family

currently includes ten members: MKP-1 (DUSP1), MKP-2 (DUSP4), MKP-3

(DUSP6), MKP-4 (DUSP9), MKP-5 (DUSP10), MKP-7 (DUSP16), MKP-X

(DUSP7), PAC1 (DUSP2), hVH3 (DUSP5), M3/6 (DUSP8) and MK-STYX

(DUSP24) (138). The catalytic sites of these enzymes feature common structural

elements such a C-terminal catalytic domain that displays homology to prototypic

VH-1 DUSP1 encoded by the vaccinia virus and an N-terminal non catalytic

domain. This non-catalytic domain of DUSPs contains a cluster of conserved

amino acid residues that function to recognize MAP kinases, known as the

kinase interaction motif (KIM) (137).

MAP kinase phosphatases are classified into three categories, dependent

upon their sub-cellular localization. The first group encompasses MKP-1, MKP-

2, PAC-1, and hVH3, which are present predominantly in the nucleus and are

encoded by highly regulated genes and are activated in response to stress

and/or mitogenic stimuli at the level of transcription (139). This group of

phosphatases preferentially dephosphorylates ERK1/2 but also has broad

substrate specificity for other MAPK’s, such as JNK and p38. A second group of

dual-specificity phosphatases is composed of MKP-3, MKP-4 and MKP-X which

are predominantly expressed in the cytoplasm and exclusively dephosphorylate

61

ERK1/2. The members of the third group are MKP-5, MKP-7 and hVH5, which

are expressed in both the cytoplasm and the nucleus and selectively

dephosphorylate the stress-activated MAPKs such as p38 and JNK, with poor

selectivity for ERK’s.

MAP kinases phosphatases are implicated in various physiological

processes, such as cytokine biosynthesis, anti-inflammatory responses, fat

metabolism and cell growth. The most well-studied protein phosphatase of this

family is DUSP1/MKP-1. Gene deletion studies demonstrate that DUSP1/MKP-

1 is involved in the immune response in several types of cells (137). Moreover,

DUSP1/MKP-1 knockout mice develop severe hypotension and multiple organ

failure and exhibit a remarkable increase in mortality. DUSP1/MKP1 is an

essential feedback regulator of the innate immune response and plays a critical

role in suppressing endotoxin shock (140). DUSP1/MKP-1 is an inducible

nuclear protein that is implicated in cell cycle control and phosphorylation (141)

and is activated at the transcriptional level in response to factors that stimulate

the expression of ERK1/2 (142). DUSP1/MKP-1 is regulated by ERK1/2 in a

negative feedback mechanism to inhibit kinase activity when the MAP kinases

are activated. DUSP1/MKP-1 is polyubiquitinated and a target for proteosomal

degradation (143).

DUSP1/MKP-1 dephosphorylates both threonine and tyrosine residues

which deactivate the MAP kinase pathways. Therefore, expression of DUSP1

62

may represent a mechanism for an arrest of cell proliferation and survival since

ERK stimulates these events. Over expression of DUSP1/MKP-1 in vascular

smooth muscle cells resulted in inactivation of MAP kinases including ERK’s and

led to the inhibition of DNA synthesis (144). In a another study, treatment with

sildenafil inhibited proliferation of pulmonary smooth muscle cells which was

associated with a reduction in MAP kinase activity and an up-regulation in

DUSP1/MKP-1 mediated through cyclic GMP or a cyclic GMP-dependent kinase

I alpha (144). In the same study, pre-treatment with vanadate, a phosphatase

inhibitor, reversed the sildenafil-mediated inhibition of ERK phosphorylation and

inhibited the anti-proliferative effects of sildenafil (144). DUSP1/MKP1 has

physiological roles directly relevant to the regulation of carcinogenesis and may

serve as a potential target in the treatment of different types of cancer.

Basis for the Hypothesis that Ang-(1-7) Reduces Tumor Growth and Tumor Angiogenesis

Treatment with Ang-(1-7) demonstrated the capacity of this peptide to inhibit

proliferation in vitro and in vivo of VSMCs (20;30), cardiac fibroblasts (35), and

cardiac myocytes growth (35). Infusion of Ang-(1-7) into rats caused the

inhibition of neointimal growth after injury (20;28;29;145) to improve cardiac

function (21;22). In a model of wound healing, administration of Ang-(1-7)

inhibited angiogenesis stimulated by Ang II (25;31). Together, these studies

demonstrate the anti-growth and anti-angiogenic effects of this heptapeptide on

actively proliferating cells. Epidemiological studies showed that treatment of

hypertensive patients with ACE inhibitors reduced the risk of cancer, especially

63

gender related cancers such as breast cancer (52). Important to this study, ACE

inhibitors elevate the anti-proliferative peptide Ang-(1-7) and decrease the

growth-promoting peptide Ang II. Moreover Ang-(1-7) inhibited the proliferation

of lung cancer cells mediated by an AT(1-7) receptor and reduced the size and

growth of human lung tumors in a xenograft model of athymic mice (146). Based

on these studies, we proposed that Ang-(1-7) would inhibit triple-negative

breast cancer cell growth mediated by a unique AT(1-7) receptor present in

these cell types and that the heptapeptide may serve as a novel targeted

therapy for the treatment of triple-negative breast cancer.

64

CHAPTER II MATERIALS AND METHODS

Materials: Ang-(1-7) and [D-Ala7]-Ang-(1-7) were purchased from Bachem (King

of Prussia, PA), while [D-Pro7]-Ang-(1-7) was synthesized by GenScript

Corporation (Piscataway, NJ). Dulbecco’s modified Eagle’s medium (DMEM),

Opt-MEM, Hams F12, penicillin, RPMI-1640, streptomycin, fetal bovine serum

(FBS), and Hypoxanthine-Aminopterin-Thymidine (HAT) supplement were

obtained from Gibco Invitrogen BRL (Carlsbad, CA). Matrigel was purchased

from BD Biosciences (Bedford, MA). TRIzol reagent was obtained from Life

Technologies/Invitrogen (Carlsbad, CA). RQ1 DNase was purchased from

Promega (Madison, WI) while the Taqman Universal PCR Master Mix and the

VEGF and DUSP1 primer probe sets were obtained from Applied Biosystems

(Foster City, CA). siRNA’s to DUSP1 and cyclophillin were designed and

purchased from Dharmacon (Chicago, IL). TEMED ((CH3)2NCH2CH2N(CH3)2

was obtained from Sigma-Aldrich, St. Louis, MO). Ammonium Peroxysulfate

((NH4)2S2O8 was purchased from Fisher Scientific, New Jersey, USA). Tween-20

was obtained from Fisher Scientific (New Jersey, USA). Antibodies were

obtained from the following sources: Ki67 and CD34 (Abcam, Cambridge, MA),

VEGF (Santa Cruz Biotechnology, Santa Cruz, California), PlGF (Novus

Biologicals, Littleton, CO), DUSP1 (Upstate, Lake Placid, NY), ERK1/ERK2-

P44/42 kinases and MEK 1/2 dual specificity kinases (Cell Signaling

Technology, Danvers, MA), actin (Sigma-Aldrich, St. Louis, MO), EF1α (Upstate,

Temecula, CA), and polyclonal and HRP-conjugated secondary antibodies (GE

Health care, Buckinghamshire, UK).

65

Cell lines: The MDA-MB-231 (HTB-26) cell line originated from an

adenocarcinoma of the the mammary gland and is of epithelial cell type, derived

from a pleural effusion of a 51 year old Caucasian female. The HCC38 (CRL-

2314) originated from a ductal carcinoma of the mammary gland and is of

epithelial cell type, derived from a 50 year old Caucasian female. These breast

cancer cell lines do not express the estrogen or progesterone receptor and have

basal expression of the epidermal growth factor receptor HER2. The A549

(CCL-185) human lung carcinoma cell line is derived from a 58 year old

Caucasian male. The MDA-MB-231, HCC38 and A549 cell lines were purchased

from the American Tissue Culture Collection (Manassas, VA). Human Umbilical

Vein Endothelial Cells (HUVECs) where purchased from Lonza (Walkersville,

MD). EA.hy926 endothelial cell line, derived by fusing human umbilical vein

endothelial cells with the permanent human cell line A549, were provided by Dr.

C.J. Edgell from the Department of Pathology and Laboratory Medicine at the

University of North Carolina at Chapel Hill.

Cell Culture: MDA-MB-231 breast cancer cells were grown in Dulbecco’s

Modified Eagles Media (DMEM) containing 10% FBS, 100 µg/mL penicillin, 100

units/mL streptomycin and 10 nM Hepes (pH = 7.2). HCC38 cells were grown in

RPMI-1640 Medium containing 10% FBS, 100 µg/mL penicillin, 100 units/mL

streptomycin and 10 nM Hepes pH 7.2). HUVECs were maintained in

endothelial cell growth medium 2 (EGM-2, Lonza, Walkersville, MD) containing

66

growth factors R3-IGF, rhVEGF, rhFGF-B, rhEGF, 10 % FBS, 50 µg/ml

gentamicin, and 50 µg/ml amphotericin. EA.hy926 cells were maintained in

DMEM medium with 10% FBS, 100 μg/mL penicillin, 100 units/mL streptomycin

and HAT supplement. A549 human lung adenocarcinoma cells were grown in

Ham’s F12 medium with 10% FBS, 100 μg/mL penicillin, and 100 units/mL

streptomycin. Cells were grown at 37°C in a humidified atmosphere of 5% CO2

and 95% room-air. Cells were made quiescent by treatment for 24 h with serum-

deficient (deficient = reduced serum or deleted, with no serum) media.

Effects of Ang-(1-7) on in vitro Breast Cancer Cell Growth

Cell Number: Confluent cell monolayers from the MDA-MB-231 and HCC 38

cell lines were grown in T75 tissue culture flasks. Cell monolayers were washed

with PBS [50 mmol/L NaPO4 (pH 7.2), 100 mmol/L NaCl] and removed with

trypsin/EDTA (0.05% Trypsin / 53 mM EDTA) The contents of a T75 flask were

diluted 1:5 and cells were seeded into individual 24-well tissue culture plates in

the presence of 1% FBS. On the first day after plating and every 24 h

afterwards, half of the cells were treated with 100 nM Ang-(1-7), added daily due

to degradation of the heptapeptide. On days 1, 3, 6, and 9, cells were removed

using trypsin/EDTA and were counted using a hemocytometer to measure cell

number. The total number of cells were counted within four grid areas of 1 mm2

(divided into 16 squares of 0.0625 mm2 ) in the hemocytometer. Cell number/mL

was calculated as the (total cells/4) x 10,000 * dilution factor.

67

Transfection of siRNA to DUSP1: siRNAs duplexes that targeted mRNA

sequences encoded by DUSP1 were synthesized by Dharmacon (5'-

CCAAUUGUCCCAACAUUU-3' 5’-GCAUAACUGCCUUGAUCAA-3’ 5’-

GCGCAAUCUUCUUCCUCA-3’ 5’-GAAGGGUGUUUGUCCACUG-3’). MDA-

MB-231 breast cancer cells were plated in 100 mm Petri dishes. Once cultures

reached 50 to 60% confluence, the media was replaced with OptiMEM (source in

materials) media with 10% FBS. siRNAs were delivered using a solution of

Oligofectamine (source in materials) with siRNAs against DUSP1 or cyclophilin

as a control at a final concentration of 0.24 µM. Cyclophilin has no known

homology to any gene sequences in mice, rats or humans; siRNAs to cyclophilin

are used to determine non-specific effects of transfection. Cells were incubated

with siRNA’s for 48 h; at the end of this period, cells were made quiescent by

treatment with OptiMem serum-deficient media for 24 h. Quiescent cells were

treated with 100 nM Ang-(1-7) for 4 h. DUSP1 expression was determined with

the use of a specific antibody, as described in the Western blot hybridization

section below.

Effects of Ang-(1-7) on in vitro Endothelial Tubule Formation and in vivo Angiogenesis

Endothelial Cell Tubule Formation Assay. The wells of a cooled 96-well plate

were coated with 50 μL of Matrigel and EA.hy926 cells were seeded onto the

polymerized matrix at a density of 1 x 104 cells/well to visualize tubule formation.

68

Cells were treated with Ang-(1-7) at increasing concentrations (10-12 - 10-6 M) to

determine dose-dependency. Cells were incubated with Ang-(1-7) at a

concentration of 100 nM in the presence or absence of 1 µM [D-Pro7]-Ang-(1-7),

to demonstrate the effect of dose and to assess whether the Ang-(1-7) response

was receptor-mediated. A control group which had no treatment was also

included in the experiments. After 16 h, tubule formation was assessed under a

light microscope and quantified by counting the number of tubules. Experiments

were performed in triplicate and data is expressed as the average number of

tubules per assay for receptor identification and as percent from control to

determine dose-dependence.

Measurement of Thymidine Incorporation Into Endothelial Cells: EA.hy926

cells were grown on 24-well culture dishes in media containing 10% FBS. Cells

were made quiescent by a 48-h incubation in serum-free media. Cells were

stimulated with 1% FBS and treated with increasing concentrations of Ang-(1-7)

(10-12-10-6 M). After 48 h, [3H]-thymidine incorporation was measured as an

indication of cell proliferation. During the last 4 h of treatment, 0.25 µCi of [3H]-

thymidine/ml culture medium was added to the growth media. Cell monolayers

were washed with PBS and treated for 30 min with ice-cold 10% trichloroacetic

acid. The acid-insoluble material was dissolved in 0.2% SDS in 0.1 N NaOH and

the amount of [3H-thymidine incorporation was determined by liquid scintillation

spectrometry in the presence of 5 ml of Ecolume (ICN Biomedicals, Aurora, OH).

69

The percent of inhibition of DNA synthesis by Ang-(1-7) was calculated based

upon the control in the presence of serum alone.

Effects of Ang-(1-7) on Endothelial Cell Migration: EA.hy926 endothelial cells

were plated on 100 mm petri dishes and maintained as described above.

Confluent cell cultures were treated with serum-deficient medium for 24 h.

Confluent cell monolayers were scratched with a 20 – 200 μL pipette tip to cause

a wound and induce cell migration. Cells were stimulated with media and 10%

FBS in the presence or absence of Ang-(1-7). After 24 h treatment, cells were

examined under light microscopy and cells that migrated to the wounded area

were visualized.

Effects of Ang-(1-7) on in vivo Angiogenesis - Chorioallantoic Membrane

(CAM) Assay. Chicken eggs obtained from a local farm (Tyson Farms,

Wilkesboro, NC) were incubated in a humidified atmosphere at 37°C. At day

four, the air cell of the egg shell was removed and covered with paraffin. After

two days of incubation, 7 mm methylcellulose disks containing saline, 100 nM

Ang-(1-7), 100 nM Ang-(1-7) and 1.0 µM AT(1-7) receptor antagonist [D-Pro7]-Ang-

(1-7) (D-Pro), or 1.0 µM [D-Pro7]-Ang-(1-7) were placed onto the surface of the

CAM in an avascular area. Neovascularization was examined 24 h later by

counting branch points in pictures taken with a Nikon D200 (Melville, NY) with a

105 microlens. Data are expressed as the average numbers of branch points in

a 7 mm2 treated area.

70

Another set of experiments was performed by treating the whole embryo

with Ang-(1-7). Fertilized eggs were incubated in a humidified atmosphere at a

temperature of 37°C for 4 days. At day four, the eggs were removed from the

incubator and the shell was sterilized by applying betadine with clinical gauze. A

small incision was made in the shell using forceps and 3 mL of albumin were

removed using a sterilized syringe. The air cell of the egg shell was removed

and 3 mL of DMEM media + 1% albumin with or without 100 nM Ang-(1-7) was

added to different groups of eggs. Eggs were covered with paraffin and, on day

6 of development, neovascularization was examined by counting branch points

observed by comparing pictures taken at day 4 and day 6 with a Sony Powershot

camera DSC-F707. Data are expressed as the average of number of new

vessels per field.

Effect of Ang-(1-7) on the Growth of Human Lung Tumors

Xenograft Model of Lung Cancer. A549 cells were grown in 100 mm dishes to

~75% confluency. Cells were collected in PBS by scraping with a rubber

spatula. Cells were counted using a hemocytometer as described above. A

volume of 1.0 x 106 cells was suspended in Matrigel (50:50) and injected into the

lower flank of 10 male athymic mice (15-20 g, 4 weeks of age. Charles River

Laboratory, Wilmington, MA), to induce tumor growth. Tumor lengths were

measured twice a week during the treatment period using a caliper and tumor

volume was calculated using the formula for a semi-ellipsoid (4/3πr3)/2). After

71

the tumors reached approximately 100 mm3, the mice were placed into two

groups at random and the animals received subcutaneous injections of either

saline or 1000 µg/kg/day Ang-(1-7) in saline. The injections were administered

daily for 5 days followed by a two day rest period for 6 cycles. Mice were housed

in cages with HEPA-filtered air (12-h light dark cycle) and ad libitum access to

food and autoclaved water. The mice were anesthetized with halothane on day

42 and euthanized by decapitation. Trunk blood was collected for Ang-(1-7)

peptide measurement. Tumors were excised from animals. Half of each tumor

was flash frozen in liquid nitrogen and stored at -80°C and the other portion was

fixed in 4% paraformaldehyde for 24 h and incubated 48 h in 70% ethanol prior to

paraffin embedding. All procedures complied with the policies of the Wake

Forest University Animal Care and Use Committee.

Effect of Ang-(1-7) on the Growth of Human Triple negative Breast Cancer Tumors

Orthotopic Model of Human Breast Cancer: Ten female athymic mice (15-20

g, 4-6 weeks of age; Charles River Laboratory,) were housed in cages with

HEPA-filtered air (12-h light dark cycle) and ad libitum access to food and

autoclaved water. All procedures complied with the policies of the Wake Forest

University Animal Care and Use Committee. MDA-MB-231 cells were grown in

100 mm dishes; once cells were 75% confluent, monolayers were washed,

physically dissociated from the tissue culute dish and collected in PBS. Cells

were suspended in 50% PBS and 50% Matrigel and 2 x106 cells were implanted

72

into the mammary fat pad of 4 to 6 week old athymic female mice. Tumor size

was measured every other day using a caliper and tumor volume was calculated

using the formula V= [(4πr3/3)/2]. When tumors reached an approximate volume

of 100 mm3, mice were injected subcutaneously with either saline or 1000 μg/kg

of Ang-(1-7) every 12 h. After 30 days of treatment, animals were anesthetized

with halothane (or isofluorane) and sacrificed by decapitation. Trunk blood was

collected for Ang-(1-7) peptide measurements. Tumors were excised; half of

each tumor was flash frozen in liquid nitrogen and stored at -80˚C while the other

half of each lung was fixed in 4% paraformaldehyde for 24 h and incubated in

70% ethanol for 48 h prior to paraffin embedding.

Angiotensin Peptide Measurement: Trunk blood was collected into chilled

Vacutainer blood collection tubes (Becton-Dickinson, Franklin Lakes, NJ)

containing 25 mmol/L EDTA and a cocktail of peptidase inhibitors (0.44 mmol/L

o-phenanthrolene, 0.12 mmol/L pepstatin A, 1 mmol/L 4-chloromercuribenzoic

acid and 3 µM acetyl-His-Pro-Phe-Val-statine-Leu-Phe, a specific rat renin

inhibitor). After 20 min on ice, blood samples were subjected to centrifugation at

3,000 rpm for 20 min and plasma was withdrawn without disturbing the packed

cells. Aliquots of plasma were stored at 80°C. The immunoreactive plasma

concentration of Ang-(1-7) was measured by radioimmunoassay (RIA). The Ang-

(1-7) RIA fully recognizes Ang-(1-7) and Ang-(2-7), but cross-reacts less than

0.01% with Ang-(3-7), Ang II, Ang I, and their fragments. The RIA measures

Ang-(1-7) through competition with 125I-labeled Ang-(1-7) for antibody binding

73

sites. A secondary antibody allows separation. The bound complex is quantified

in a gamma spectrometer and the amount of Ang-(1-7) is determined by

comparison to a series of standards that are assayed simultaneously. The

assays were conducted in the Radioimmunoassay Core of the Hypertension

Center at WFUSM.

Effect of Ang-(1-7) on Tumor Cell Proliferation and in Endothelial Tumor Vessel Density.

Immunohistochemistry: Orthotopic breast tumors and lung tumor xenografts

were fixed in 4% paraformaldehyde for 24 h and incubated in 70% ethanol for 48

h prior to embedding in paraffin. The embedded tumors were cut into five micron

thick sections and stained with hematoxyline & eosin (H&E) to determine

morphology. Immunostaining with specific antibodies to Ki67 and CD34 was

used to measure cell proliferation and vessel density, respectively. Tumor

sections were deparaffinized, rehydrated and placed in distilled water for 5 min.

Antigen retrieval was performed by incubating tissue sections in 10 mM citrate

buffer, pH 6.0, for 35 min in a vegetable steamer. The slides were cooled for 20

min, placed in distilled water for 5 min, and then incubated overnight with the

primary antibodies at 4ºC. Slides were washed 5 times with 1X Tris buffer (0.1M

M Tris-HCl, pH = 8.2-8.4) containing 0.5% casein to prevent non-specific

binding. A secondary biotinylated antibody was added subsequently for 30 min

at 32ºC. Slides were washed 5 times in Tris buffer followed by a 1:20 dilution of

Streptavidin-alkaline phosphatase conjugate for 30 min at 32ºC. Slides were

74

washed a second time in in Tris buffer and incubated with Vector Red substrate

for 5 min. The slides were then washed in 2 times in 0.1 M Tris-HCl buffer (pH

8.2 - 8.4) and 5 times with distilled water. The sections were counterstained with

Mayer’s hematoxylin for 5 min and washed under running water for 5 min. Slides

were dehydrated through a series of graded alcohols and cleared using p-

xylene. Stained sections were visualized with a Leica DM 4000 (Leica

Microsystems, Bannockburn, IL) microscope and photographed with QImaging

Retiga 1300R camera (QImaging Co. Surey, BC, Canada). A computer-assisted

counting technique with a grid filter to select cells was used to quantify the

immunohistochemical staining of Ki67. The positive cells were expressed as

percentage of the total cell number examined (100 cells sampled from each

tissue site within a tumor section). Endothelial vessel density was examined in

tumor tissue sections scanned at low power magnification (50x) with a Leica DM

microscope to identify areas of the tumor with the highest density (“hot spots”) of

CD34 positive stained vessels. Selected sections were counted without the

knowledge of treatment group at a 200x magnification in a 0.3 mm2 field using

the Simple PCI Version 6.0 computer-assisted imaging software (Hamamatsu

Corporation, Sewickley, PA.) and photographed with the QImaging Retiga

1300RCamera. The number of immuno-positive vessels, as defined by Weidner

et al., (147) was expressed as the average of 6 fields (0.3 mm2) selected per

tumor.

Effect of Ang-(1-7) on Tumor Gene Expression of VEGF, PlGF, DUSP1, COX-2, PGES.

75

RNA Isolation: RNA was isolated from human breast orthotopic tumors, human

lung tumor xenografts, cultured MDA-MB-231 breast cancer cells or A549 lung

cancer cells using TRIzol (Life Technologies/Invitrogen, Carlsbad, CA), according

to the manufacturer’s instructions. Media was removed from 100 mm Petri

dishes and 3 mL TRIzol reagent was added to the cell monolayer. The total cell

lysate was scraped and transferred to sterile tubes containing two stainless steel

beads. For tumor tissue, approximately 100 mg of tissue was cut and placed in a

2.0 mL tube; 1 mL TRIzol was added with two stainless steel beads. Samples

were homogenized using the TissueLyzer (Retsc, Hann, Germany) at 25 X

speed for 1 min. Cellular debris was removed by centrifugation at 2,500 x g for

10 min. The resultant supernatant was transferred to a clean 1.5 mL tube before

proceeding with RNA isolation. Chloroform (200 µLper mL of TRIzol) was added

to the resultant supernatant. The mixtures were shaken vigorously for 20 s and

allowed to sit at room temperature for 2 min. Samples were subjected to

centrifugation for 30 min at 4,000 x g at 4ºC. The aqueous layer was transferred

to a 1.5 mL tube and 500 µL of isopropanol per 1 mL of TRIzol was added;

samples were incubated for 10 min at RT. Samples were mixed using a vortex

and subjected to centrifugation for 10 min at 3,000 x g at 4ºC. The supernatants

were discarded and the pellets were allowed to dry before dissolving in nuclease-

free water (Gibco Invitrogen BRL, Grand Island ,NY). RNA concentration was

determined by diluting samples 1:100 in Milli-Q water. Diluted samples were

76

quantified in a Biorad Smartspec 3000 (Hercules, CA) using the adsorption of

light at 260 and 280 nm wavelength.

RQ1 RNase-Free DNase Reaction: The RNA was incubated with RQ1 DNase

to eliminate any residual DNA that would amplify during the PCR. Enzyme, RNA

sample, water and RQ1 RNase-Free DNase 10x Reaction buffer were

combined;1 unit (1 µL) of enzyme was used per 1 µg of RNA, 1 µL of Buffer was

added per 10 µl of total reaction volume, and nuclease-free water was added to a

final volume of 10 µL. The reaction was incubated at 37ºC in a water bath for 30

min. After this incubation period, the reaction solution was brought to 200 µL in

total volume with nuclease-free water. Phenol and chloroform (100 µL each)

were added to the mixture and the reaction was mixed using a vortex for at least

10 s. The samples were subjected to centrifugation at 12,000 xg for 10 min. The

top phase was transferred to a new tube and 1/10 of the volume of 5 M

ammonium acetate (Sigma-Aldrich, St. Louis, MO) was added to the reaction

(about 20 µL) and 2.5 times the total volume of 100% Ethanol (EtOH, C2H5OH)

was added. The solution was incubated for at least 2 h or overnight at -20 ˚C.

The reaction mixture was subjected to centrifugation at 12,000 xg for 15 min.

The EtOH layer was removed and 0.5 mL of 75% ETOH was added prior to an

additional centrifugation at 7,500 xg for 10 min. ETOH layer was removed and

the pellet was air dried. The pellet was re-suspended in at least 30 uL of RNase-

free H20 and incubated for 10 min at 55 ºC.

77

Determination of RNA integrity: The RNA concentration and integrity were

assessed with an Agilent 2100 Bioanalyzer using an RNA 6000 Nano LabChip

(Agilent Technologies, Palo Alto, CA). RNA samples and RNA ladder were

denatured at 70˚C for 2 min. Gel reagents were allowed to equilibrate at room

temperature for 30 min prior to gel preparation. RNA 6000 gel matrix (550 µL)

was added into a spin filter. This was subjected to centrifugation for 10 min at

1500 xg and 65 µL were aliquoted into a 0.5 mL RNase-free microfuge tube. The

RNA 6000 nano dye was mixed by vortex for 10 s and 1 µL of this dye was

added to the aliquoted gel matrix. The solution was mixed by vortex and

subjected to centrifugation at 13000 xg for 10 min at room temperature. The gel

was loaded into the RNA chip placed in the priming station and 9.0 µL of the gel-

dye mix was added into a designated well. A plunger was used to spread the gel

onto the chip, which was held and released after 30 s. Another 9.0 µL of the gel-

dye mix was added to the designated well and solution was spread onto the chip

as explained above. RNA 6000 nano marker (5 µL) was added to the wells.

Sample (1 µL) was loaded into respective wells. The chip was placed in an

adapter that mixes by vortex at a speed of 2400 rpm. The chip was placed in the

bioanalyzer and the samples were analyzed using the 2100 expert software.

This software expresses the data as an Electropherogram. If the sample

contains intact RNA, the sample will be resolved into three peaks corresponding

to the RNA marker, the 18s rRNA and 28s rRNA peaks.

78

Reverse Transcription: RNA samples were denatured at 70˚C for 5 min and

placed on ice for at least 5 min. The samples were collected by centrifugation at

low speed, to collect the liquid in the bottom of the tube. Approximately 1 µg of

total RNA was reverse transcribed using avian myeloblastosis virus (AMV) RT in

a 20 µL reaction mixture containing the reagents expressed in the following table:

79

Table II:

Preparation of Master Mix for Reverse Transcriptase Reaction

Component Volume/Reaction (µL) Volume/Master Mix

5% AMV Buffer 4.0 4.0 x # of Samples

dNTP’s (10mM) 2.00 2.0 x # of Samples

RNAsin (2,500 units) 0.5 0.5 x # of Samples

Sodium Pyrophosphate (40 mM)

2.0 2.0 x # of Samples

AMV (1,000 units) 1.5 1.5 x # of Samples

Total 10.0

80

The reaction solution was mixed by inverting the tube and 10 µL of the

master mix was added to denatured RNA. Samples were placed in the Peltier

thermal cycler (MJ Research Ramsey, Minnesota). The reaction was performed

at 37˚C for 60 min, followed by 95 ˚C for 5 min to terminate the reaction; the

thermocycler cooled to 10˚C to hold samples until removal.

Real-Time PCR: Two microliters of cDNA were added to TaqMan Universal

PCR Master Mix (Roche, New Jersey, USA) with 1 µL per reaction of COX-2,

PGIS, PlGF, VEGF, or DUSP1, 18S rRNA-specific primer/probe sets and

amplification was performed on an ABI 7000 Sequence Detection System

(Applied Biosystems, Foster City, CA). The mixtures were heated at 50°C for 2

min and at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1

min. All reactions were performed in triplicate and 18S rRNA served as an

internal control. The results were quantified as Ct values, where Ct is defined as

the threshold cycle of PCR at which the amplified product is first detected, and

defined as relative gene expression (the ratio of target/control).

Western Blotting to analyze the effect of Ang-(1-7) on VEGF, PlGF, MAP Kinase, and DUSP1 protein expression in human tumors.

Preparation of Protein Extracts: Frozen tumor tissue was cut into ~ 1 to 2 mm2

size pieces and was transferred to a 2 mL Eppendorf tube with one stainless

steel bead and 1 mL of PBS. Tumor samples were then homogenized using a

81

Quiagen TissueLyser at a frequency of 25 1/s for 1 min. The homogenate was

transferred to a clean tube and was solubilized by adding boiling 3% SDS-10% β-

mercaptoethanol. Cellular protein from breast cancer cells of the MDA-MB-231

cell line that were ~75% confluent was obtained by washing confluent

monolayers of cells growing in 100 mm Petri dishes with PBS containing 0.01

mM NaVO4 to prevent the dephosphorylation of activated, phosphorylated

proteins in the samples. The monolayers were solubilized in lysis buffer (100

mmol/l NaCl, 50 mmol/l NaF, and 5 mmol/l EDTA, 1% Triton X-100, and 50

mmol/l Tris·HCl, pH 7.4 containing 0.01 mmol/l NaVO4, 0.1 mmol/l PMSF, and

0.6 µmol/l leupeptin) by incubating on an ice-water slurry for 25 min. Protein was

measured by a modification of the Lowry method, as described below (148).

SDS-PAGE Gel electrophoresis: Ten or 15% SDS-PAGE gels (running gel)

were prepared using a solution of 30% acrylamide, 1.5 M Tris-HCl, pH 8.8, Milli

Q water, 10% SDS, TEMED and 10% Ammonium perodoxysylfate (APS). The

solution was poured into the gel glass cassette and allowed to polymerize in the

Mini-Protean 3 Electrophoresis Module Assembly from Biorad (Hercules, CA). A

stacking gel solution was prepared containing 3% acrylamide, 0.5 M Tris-HCl pH

6.8, Milli-Q water, 10% filtered SDS and 10% APS. The solution was poured

over the running gel and the appropriate size comb was placed between gel

glass cassette spacers. The stacking gel was allowed to polymerize. Samples

containing an equal amount of protein were loaded onto individual wells of the

gel and proteins were separated by electrophoresis at constant current of 200 V.

82

Transfer of Proteins to PVDF membranes: Following electrophoresis, gels

were allowed to equilibrate in transfer buffer (25 mM Tris-HCl, 192 mM Glycine,

pH = 8.3 and 20% methanol) for at least 15 min. After this period, the transfer

sandwich was assembled, with all the components pre-soaked in transfer buffer.

The gel cassette holder was placed with gray side down with a fiber pad placed

on the gray side of the gel cassette holder. This was followed by a sheet of filter

paper, the equilibrated gel, a sheet of polyvinylidene diflouride membrane

(PVDF) (Hybond-P Amersham Biosciences, Arlington Heights, IL), a second

layer of filter paper and a fiber pad. The assembled gel sandwich was placed

into the electrode module. Proteins were transferred to hydrophobic PVDF

membrane by electroblotting in transfer buffer for one hour at constant current of

100 V in a Mini-Transfer Blot Electrophoretic Transfer Cell, (Biorad, Hercules

CA).

Immunoblotting: The PVDF membranes were incubated in Blotto [5%

evaporated milk, 0.1% Triton X-100 in Tris-buffered saline (TBS; 50 mM Tris-HCl,

pH 7.4, 50 mM NaCl]] for one h, to block non-specific binding. The membranes

were probed with primary antibodies specific to DUSP1 (at a dilution of 1:2500),

extracellular signal-regulated kinase (ERK1/ERK2) (P44/42 kinases, at a 1:2500

dilution); PlGF (at a 1:1000 dilution), and VEGF (at a 1:10,000 dilution), overnight

at 4 ˚C. This was followed by incubation with polyclonal horseradish peroxidase

83

(HRP)-conjugated secondary antibodies (GE Health care, Buckinghamshire, UK)

for 1 h at room temperature.

Quantitation of Immunoblots: Immunoreactive products were visualized by

mixing chemiluminescence reagents from SuperSignal Femto or Pico West

(Pierce Biotechnology, Rockford, IL) 1:1 and spreading the mixture over the

blotted PVDF membrane. The immunoreactive products were quantified by

densitometry using the MCID digital densitometry software (Cambridge, UK).

The optical densities of the immunoreactive samples were evaluated by

comparing products of cells treated with serum alone to cells treated with Ang-(1-

7) and serum. Protein loading was visualized by incubation of stripped

membranes (explained below) with a monoclonal antibody to actin (dilution) or

EF1α (dilution) overnight at 4˚, followed by incubation with monoclonal

horseradish peroxidase (HRP)-conjugated secondary antibodies for one h;

protein density was analyzed as explained above.

Stripping Western Blot Membranes: Membranes are washed in TBS and

Tween twice for 15 min, prior to stripping. The membranes were incubated with

stripping buffer [62.5 mM Tris-HCl (pH 6.8) and 0.1 M 2-Mecaptoethanol] for 30

min with constant shaking in a 50°C water bath. After this period, the

membranes were washed twice in TBS and Tween for 15 min and re-blocked

with Blotto. The re-probing procedure was followed in the same manner as

specified in the immunoblotting section above. Alternatively, the membranes

84

were stripped using the Western Blot Recycling Kit (Alpha Diagnoistic

International, San Antonio, TX). The membrane was washed initially as

expressed above and incubated with the diluted 10X antibody stripping solution,

by constant shaking for 30 min at room temperature. After this period, the

membrane was incubated in a 1:20 diluted 20X milk-based blocking buffer for 5

min and reprobed as explained in the immunoblotting section above.

Lowry Protein Assay: To determine protein concentration, we used the

modified Lowry method (148). Protein lysates (unknowns) or Bovine Serum

Albumin (BSA) standards were combined with Milli-Q water in 12 x 75 glass

tubes. The standards were added from a concentration of 8.44 µg/mL to 54.4

µg/mL to create a standard curve in a total volume of 100 uL, as described in the

table III.

85

Table III:

Lowry Protein Assay Standard Curve.

Standard µg of BSA µL BSA µL H2O

1 0.00 0 100

2 8.16 15 85

3 10.88 20 80

4 16.32 30 70

5 21.76 40 60

6 32.64 60 40

7 43.52 80 20

8 54.40 100 0

86

A 5 to 10 uL aliquot of the protein lysates was diluted in 95 to 90 µL of

water; this volume was estimated to be in the range of the concentrations of the

standard curve. One mL of Reagent 1 (10% perchloric acid, 1%

Phosphotungstic acid) was added to all the samples and the standards. The

samples were incubated in an ice bath for 1 h. At the end of this period, the

samples were subjected to centrifugation at 5000 rpm for 5 min. The

supernatant was decanted and tubes were blotted dry to remove any remaining

solution. Reagent 2 (2% Na2CO3 in 0.1 N NaOH), Reagent 3 (1% CuSO4-5H2O

Copper sulfate), and Reagent 4 (2% Sodium-Potassium Tartarate) were mixed,

with 1 mL of each reagent 3 and 4 added per 100 mL of reagent 2 to make

Reagent 5. One mL of Reagent 5 was added to each tube and samples were

incubated for 10 min at room temperature. One hundred (100 µL) of a 1:1

solution of H2O and 2 N Folin-Ciocalteu (Sigma-Aldrich, St.Louis, MO) reagent

(Reagent 6) was added to each tumbe while mixing using a vortex. The tubes

were incubated for an additional 30 min. Absorbance was measured at 750 nm

using samples containing no protein as a blank. A standard curve was

generated and the concentration of each sample was determined by comparison

to the standard curve, using the Lowry Program Software G.A. McPherson 1985

(Elsevier Biosoft, Cambridge, UK).

Statistics. All data are presented as the mean ± standard error of the mean

(SEM). Statistical differences were evaluated by Student’s t test or one way

87

analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The criterion

for statistical significance was set at p < 0.05.

88

CHAPTER III RESULTS

Ang-(1-7) Inhibits the Growth of Triple-negative Breast Cancer Cells

The effect of Ang-(1-7) on serum-stimulated growth was examined in

human MDA-MB-231 and HCC38 breast cancer cells, as shown in Figure 9A and

9B, respectively. Both MDA-MB-231 cells (derived from an adenocarcinoma)

and HCC38 cells (derived from a ductal carcinoma) do not express the ER or the

PR or over-express HER2 (149;150) The total number of cells was determined

using a hemocytometer prior to incubation with Ang-(1-7) (day 0); the average

number of MDA-MB-231 and HCC38 cells before initiating treatment with Ang-(1-

7) was 21.5 x 103 and 36 x 103, respectively. Stimulation with 1% FBS caused a

time-dependent increase in the total number of both types of triple-negative

breast cancer cells. Treatment with 100 nM of Ang-(1-7) in the presence of 1%

FBS significantly reduced the number of cells on day 4, 6, and 8 by 59.3, 50.3

and 54.4%, respectively, in MDA-MB-231 cells, and on day 3, 6 and 9, by 46.4,

52.0 and 41.6%, respectively, in HCC38 cells. The number of cells treated in the

presence of 1% FBS and Ang-(1-7) never reached the same number of cells of

those treated with 1% FBS alone over the total time course. These results

indicate that Ang-(1-7) reduces cell growth in two different triple-negative cell

lines.

89

Figure 9: Effect of Ang-(1-7) on cell number. MDA-MB-231 (Panel A) and

HCC38 (Panel B) cells were grown in the presence of 1% FBS or 1% FBS in

combination with 100 nM Ang-(1-7). Ang-(1-7) was added on day 1 and was

refreshed each day due to degradation of the heptapeptide. The cell number

was determined on the days indicated, by counting the total cell number per well.

Triplicate wells of treated 24-well cluster plates were counted at each time point

and averaged. n = 3-4 of cells from different passage numbers, in triplicate; *

denotes p < 0.05 for cells treatment with FBS alone compared to cells treated

with Ang-(1-7) and FBS.

90

0 2 4 6 80

50

100

150

200

250FBSFBS/Ang-(1-7)

**

*

Time (days)

Num

ber o

f Cel

ls x

100

0

Panel A MDA-MB-231 cells

0 2 4 6 80

25

50

75

100

125FBSFBS/Ang-(1-7)

**

*

Time (Days)

Num

ber o

f Cel

ls x

100

0

Panel B HCC38 cells

91

Subcutaneous Injection of Ang-(1-7) Inhibits Triple-Negative Breast Cancer Tumor Volume

The effect of Ang-(1-7) on tumor growth was examined in mice bearing

orthotopic triple-negative breast tumors. Actively proliferating MDA-MB-231 cells

(2 x106 cells in PBS: Matrigel) were injected into the mammary fat pad of athymic

mice. The use of this orthotopic model allows the breast cancer cells to grow in a

clinically-relevant tumor microenvironment. Once tumors reached an

approximate volume of 100 mm3, the mice were injected subcutaneously with

either saline or 1000 μg/kg of Ang-(1-7) in saline every 12 h. The concentration

of Ang-(1-7) used for this particular study is based on dose-escalating studies in

nude mice performed in our laboratory, demonstrating that subcutaneous

administration of 1000 µg Ang-(1-7)/kg was well tolerated by mice with no

change in body weight or water consumption and resulted in a 3-4-fold increase

in circulating Ang-(1-7) [Gallagher and Tallant, unpublished observation]. The

first day of treatment was designated as day 0 and the mice were sacrificed after

30 days of treatment. Throughout the treatment period, the animals maintained

their body weight and water consumption and showed no evidence of reduced

motor function. Furthermore, no obvious pathologic abnormalities were observed

in major organs following sacrifice, indicating a lack of toxic side effects at the

dose given.

The mean tumor volume of each treatment group was similar prior to the

beginning of treatments (saline, 113.3 ± 7.0 mm3; Ang-(1-7), 107.6 ± 6.8 mm3).

During the 30 days of treatment, tumors of the saline-injected mice continued to

92

grow, whereas tumor volume was significantly reduced in mice injected with Ang-

(1-7) (Figure 10). The average volume of the tumors from mice treated with

saline was 432 ± 85.9 mm3, a 3.8-fold increase over the volume at the beginning

of the treatment period. In contrast, the tumors from mice injected with Ang-(1-7)

only grew to 69.9 ± 18.9 mm3, an 84% reduction compared to the volume of

tumors from saline-treated mice. At the conclusion of the treatment, the mice

were euthanized and the tumors were removed and weighed. As shown in

Figure 11, tumor weight was also significantly reduced by treatment with Ang-(1-

7). The tumors from mice injected with saline weighed 1.01 ± 0.21 g, while the

tumors from mice administered Ang-(1-7) weighed 0.49 ± 0.04 g (n = 5, p <

0.05),. This represents a 52% reduction in tumor weight.

Ang-(1-7) Reduces Triple-Negative Tumor Cell Proliferation

A portion of each tumor was fixed in formalin for histological analysis and

5-μm tumor sections from mice infused with saline or Ang-(1-7) were stained with

an antibody to Ki67 as a marker of proliferation. Ki67 is a nuclear protein present

in the active phases of the cell cycle and is not expressed in quiescent cells.

Cells in tumors from mice treated with saline showed abundant Ki67

immunoreactivity indicating active cell growth (Panel A, Figure 12). However,

cells in tumors from mice injected with Ang-(1-7) showed little Ki67

immunoreactivity, suggesting a lack of cell proliferation in the presence of the

heptapeptide. As shown in Panel B of Figure 12, the percentage of proliferating

cells in tumors from saline-treated mice was significantly increased when

93

compared with the percentage of proliferating cells in tumors from Ang-(1-7)-

treated mice (84.2 ± 8.2% compared to 41.0 ± 7.6%, n = 5, * denotes p < 0.05).

94

Figure 10: Inhibition of orthotopic breast tumor growth by Ang-(1-7).

Tumors from mice injected with either saline or 1000 µg/kg every 12 h were

measured by a caliper every other day and volumes were calculated using the

formula for a semi-ellipsoid: (4/3πr3)/2. Day 0 represent the first day of treatment

with either saline or Ang-(1-7). n = 5, * denotes p < 0.05 of tumors from mice

treated with Ang-(1-7) compared to tumors of mice treated with saline.

95

0 5 10 15 20 25 300

100

200

300

400

500

600 Ang-(1-7)Saline

*****

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96

Figure 11: Reduction in tumor weight by Ang-(1-7). The tumors from mice

injected with either saline or Ang-(1-7) were weighed at the time of sacrifice (day

30). n = 5, * denotes p < 0.05 of tumors from mice treated with Ang-(1-7)

compared to tumors of mice treated with saline. The insert shows representation

photographs of tumors from mice treated with saline or Ang-(1-7) for 30 days.

97

Saline Ang-(1-7)0.0

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98

Figure 12: Effect of Ang-(1-7) on tumor cell proliferation. Panel A shows

representative photomicrographs of tumor tissue sections from mice treated with

either saline or Ang-(1-7) that were analyzed immunohistochemically using

antibodies against Ki67 magnification, x200. Panel B shows quantification of

positive immunoreactive nuclei of cells stained with the Ki67 antibody. n = 5 for

each group; * denotes p < 0.05 relative to control.

99

Saline

Ang-(1-7) Saline Ang-(1-7)

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100

Effect of Ang-(1-7) on cyclooxygenase-2 and prostaglandin E synthase gene expression

Elevated expression of COX-2 and over production of prostaglandins is

observed in many human cancers (151). COX-2 is over-expressed in 40% of

breast carcinomas and correlates with an decreased disease-free survival (152).

COX-2 derived prostaglandin E2 (PGE2) can promote tumor growth by binding to

its receptors and activating signaling pathways which control cell proliferation,

migration, apoptosis, and/or angiogenesis (151). We previously reported that

treatment with Ang-(1-7) significantly reduced COX-2 mRNA and protein in

tumors of Ang-(1-7)-infused mice when compared to mice treated with saline as

well as in the parent A549 human lung cancer cells in tissue culture (146). Total

RNA was isolated from human breast cancer orthotopic tumors treated with

saline or Ang-(1-7) to determine whether the heptapeptide reduces COX-2 and

PGES mRNA in triple-negative orthotopic breast tumors. No significant

difference in COX-2 gene expression was observed between the treatment

groups [1.008 ±. 0.128 relative gene expression in tumors from saline-treated

mice as compared to 1.210 ± 0.106 in tumors from Ang-(1-7)-treated mice, n = 5

in each group] (Figure 13). Similarly, as shown in Figure 14, Ang-(1-7)

administration had no effect on PGES mRNA [PGES mean gene expression in

saline-treated tumors 1.024 ± 0.129 versus 1.016 ± 0.0869 PGES mean relative

gene expression in Ang-(1-7)-treated tumors]. This indicates that Ang-(1-7) does

not reduce COX-2 or PGES gene expression to reduce triple-negative breast

cancer growth, suggesting a different mechanism from the one reported in our

studies in lung cancer (146).

101

Figure 13: Effect of Ang-(1-7) on COX-2 gene expression in triple negative

orthotopic tumors. RNA was isolated from tumor tissue from mice injected with

saline or Ang-(1-7) and COX-2 mRNA was quantified by RT real-time PCR.

COX-2 mean gene expression in saline treated tumors 1.008 ±0.128 vs. 1.210 ±

0.106 COX-2 mean relative gene expression in Ang-(1-7) treated tumors, n = 5.

102

Saline Ang-(1-7)0.0

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103

Figure 14: Effect of Ang-(1-7) on PGES gene expression in triple negative

orthotopic tumors. mRNA was isolated from tumor tissue from mice injected

with saline or Ang-(1-7) and PGES mRNA was quantified by RT real-time PCR.

PGES mean gene expression in saline treated tumors 1.024 ± 0.129 vs. 1.016 ±

0.087 PGES mean relative gene expression in Ang-(1-7) treated tumors, n = 5.

104

Saline Ang-(1-7)0.0

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105

Ang-(1-7) reduces MAP kinase activity

Ang-(1-7) caused a significant reduction in MAP kinase activities

(ERK1/ERK2) in lung cancer cells and tumors (60;146). ERK1 and ERK2

activities were quantified in MDA-MB-231 cells and tumors, to determine whether

Ang-(1-7) reduces triple-negative breast cancer growth by attenuating MAP

kinase activities. ERK1 and ERK2 auto-phosphorylation and activation were

measured by Western blot hybridization using antibodies to phospho-

ERK1/ERK2. Ang-(1-7) significantly reduced the phosphorylation of ERK1 and

ERK2 when compared with the elevated phosphorylation expressed by the

saline-treated tumors as shown in Figure 15 [0.58 ± 11.0 ERK1 expression in

saline tumors versus 0.03 ± 0.01 expression in Ang-(1-7) tumors, n = 5, *

denotes p < 0.0008 and 0.73 ± 0.04 ERK2 expression in saline tumors vs. 0.14 ±

0.08 expression in Ang-(1-7) tumors, n = 5, * denotes p < 0.0002)]. The

reduction in MAP Kinase activity suggests that the observed reduction in

proliferation in the presence of the heptapeptide is caused by an inhibition of this

pathway.

Effect of Ang-(1-7) on MEK1/2 activity

Since Ang-(1-7) inhibited the activities of ERK1 and ERK2, we wanted to

determine the mechanisms underlying this inhibition. Therefore, we measured

the effect of Ang-(1-7) on the ERK up-stream kinases MEK1/2. MEK is a dual-

specificity kinase that phosphorylates the tyrosine and threonine residues on

ERK1 and ERK2 and is required for their activation. We measured MEK1/2

106

protein expression by Western blot hybridization using specific antibodies to

phospho-MEK1/2. As observed in Figure 16, there was no significant effect on

MEK1 and MEK2 protein expression in any of the treatment groups (0.33 ± 0.06

average protein expression in tumors from saline-treated mice as compared to

0.37 ± 0.05, n = 5 in each group). This suggest that Ang-(1-7) inhibits MAP

kinase activity by a mechanism independent of MEK1/2 inactivation.

107

Figure 15: Effect of Ang-(1-7) on MAP Kinases activity in breast orthotopic

tumors. Protein expression was analyzed by Western blot hybridization using

antibodies against the phosphorylated ERK1 and ERK2; an EF-1 alpha antibody

was used as a loading control (top panel). The data are presented as ERK1 and

ERK2 immunoreactivity (IR)/EF1α IR. n = 5, * denotes p < 0.05 relative to

treatment with saline.

108

Saline Ang-(1-7) Saline Ang-(1-7)0.00

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0.30

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0.90

**

ERK1 ERK2

ERK

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K2

Imm

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(ER

K/E

F1α

)

109

Figure 16: Effect of Ang-(1-7) on MEK1/2 activity in breast orthotopic

tumors. Protein expression was analyzed by Western blot hybridization using

antibodies against the phosphorylated MEK1/2; an EF-1 alpha antibody was

used as a loading control. The data are presented as MEK immunoreactivity

(IR)/EF-1α IR. n = 5 relative to treatment with saline.

110

Saline Ang-(1-7)0.0

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EK1/

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111

Ang-(1-7) Increases DUSP1

Dual specificity phosphatase-1 (or DUSP1) is a MAP kinase phosphatase

(MKP-1) that has affinity for tyrosine and threonine residues and causes the

specific inactivation of ERK1 and ERK2 MAP kinases (153). Ang-(1-7)

significantly increased DUSP1 protein in orthotopic triple negative breast tumors

in mice injected with Ang-(1-7) as compared to tumors from saline-treated control

animals [1.03 ± 0.16 relative protein expression in tumors from Ang-(1-7)-treated

mice as compared to 0.28 ± 0.13 in tumors from saline-treated mice, n = 5 in

each group; p < 0.002], as shown in Figure 17, Panel A. Total RNA was isolated

from tumor tissue of mice infused with saline or Ang-(1-7) and DUSP1 mRNA

was measured by RT real-time PCR to determine whether Ang-(1-7) regulates

the gene expression of this phosphatase at the level of transcription. Treatment

with Ang-(1-7) resulted in more than 175% up-regulation of DUSP1 mRNA in the

orthotopic breast tumors (Figure 17, panel B) when compared with tumors of

animals treated with saline injections (1.79 ± 0.034 versus 1.01 ± 0.07, n = 4 to 5,

* denotes p < 0.05).

The up-regulation of DUSP-1 is also dependent upon the time of

treatment. Actively growing MDA-MB-231 cells were treated with 100 nmol/L

Ang-(1-7). Total RNA was isolated from cultures at time points from 0 to 16 h

and DUSP1 gene expression was measured by RT real-time PCR as described

in the Methods Section. As shown in Figure 18, treatment with the heptapeptide

caused a time-dependent increase in DUSP1 mRNA at 4 and 8 hours after

112

treatment (0.70 ± 3.2 and 0.98 ± 4.2 relative DUSP1 gene expression at 4 and 8

h, respectively).

Ang-(1-7) regulates DUSP1 through activation of an AT(1-7) receptor

Actively growing MDA-MB-231 cells were incubated for 4 h in the presence or

absence of Ang-(1-7). As shown in panel A of Figure 19, treatment with 100 nM

Ang-(1-7) caused a 5-fold increase in DUSP1 protein expression (501.3 ± 61.9, n

= 7), suggesting that Ang-(1-7) increases protein expression in triple-negative

breast cancer cells. The up-regulation of DUSP1 in MDA-MB-231 cells by Ang-

(1-7) was blocked by 1 µM [D-Ala7]- Ang-(1-7) and [D-Pro7]-Ang-(1-7), the

selective antagonist for the Ang-(1-7) receptor (Figure 19). Treatment with both

receptor antagonists alone had no effect, such that the DUSP1 concentration

was similar to control levels. These results suggest that Ang-(1-7) up-regulates

DUSP1 protein expression through the activation of an AT(1-7) receptor.

113

Figure 17: Effect of Ang-(1-7) on DUSP1 activity in MDA-MB-231 orthotopic

breast tumors. Protein expression was analyzed by Western blot hybridization

using antibodies specific for DUSP1. The effect of Ang-(1-7) on DUSP1 was

determined by RT real-time PCR. The data are presented as relative DUSP1

gene expression in human breast cancer orthotopic tumors treated with saline or

Ang-(1-7). n = 5; *denotes p<0.05 compared to treatment with saline.

114

Panel A

Saline Ang-(1-7)0.0

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Panel B

Saline Ang-(1-7)0.00.20.40.60.81.01.21.41.61.82.0 *

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115

Figure 18. The up-regulation of DUSP1 by Ang-(1-7) is time-dependent.

The effect of Ang-(1-7) on DUSP1 mRNA was determined by RT real-time PCR.

The data are presented as relative DUSP1 gene expression in MDA-MB-231

breast cancer cells treated with 100 nM Ang-(1-7) for 16 h. n = 4, * denotes p <

0.05 relative to treatment with saline.

116

0 2 4 6 8 10 12 14 160.00

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Figure 19: Ang-(1-7) up-regulates DUSP1 protein expression by activation

of the AT(1-7) receptor. MDA-MB-231 cells were incubated for 4 h with no

addition (Basal), 1 or 100 nM Ang-(1-7), Ang-(1-7) in the presence of 1 µM [D-

Ala7]-Ang-(1-7) (D-Ala) or [D-Pro7]-Ang-(1-7), 1 µM [D-Ala7]-Ang-(1-7) or [D-

Pro7]-Ang-(1-7) alone, Ang-(1-7) in the presence of 1 µM [D-Ala7]-Ang-(1-7) (DA),

or [D-Ala7]-Ang-(1-7) alone. n = 4, * denotes p < 0.05 relative to control.

118

0

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119

Ang-(1-7) reduces tumor vessel density in triple negative breast tumors

Orthotopic triple-negative breast tumors were immunostained with the

endothelial cell marker CD34, to determine whether the reduction in lung tumor

growth observed with Ang-(1-7) administration was associated with a decrease in

tumor vessel formation. We observed numerous vessels in the periphery of the

tumor with less vessel development in the center of the tumor since the core was

characteristically necrotic. Clusters of vessels or so called vessel “hot spots”

were localized mainly in the periphery of the tumor. Sections of tumor tissue

from saline-treated animals showed strong immunoreactivity to CD34 when

compared to tumor sections from Ang-(1-7)-treated animals (Figure 20, Panel A).

As shown in Panel B of Figure 20, injection of Ang-(1-7) caused an approximate

70% reduction in tumor vessel density (average of 3 fields) when compared to

sections from saline-treated animals (87.8 ± 6.4 immunopositive CD34 cells/field

in the saline-treated group compared to 32.0 ± 7.0, n = 4, * denotes p < 0.05).

This suggests that the heptapeptide reduces angiogenesis to inhibit lung tumor

growth.

Ang-(1-7) reduces VEGF in triple-negative breast tumors

VEGF is expressed early in breast cancer carcinogenesis and is the main

pro-angiogenic factor that stimulates neovascularization from pre-existing

vessels. VEGF was measured by Western blot hybridization in total protein

homogenates isolated from MDA-MB-231 human breast cancer orthotopic

tumors to determine whether the heptapeptide reduces VEGF in vivo. Ang-(1-7)

120

significantly reduced VEGF protein in breast orthotopic tumors from mice injected

with Ang-(1-7) as compared to tumors from saline-treated control animals [0.50 ±

0.07 relative protein expression in tumors from saline-treated mice as compared

to 0.14 ± 0.07 in tumors from Ang-(1-7) treated mice, n = 4 and 5, respectively; *

denotes p < 0.0087], as shown in Figure 21, Panel A. Total RNA was isolated

from human breast cancer orthotopic tumors treated with saline or Ang-(1-7) and

VEGF mRNA was quantified by RT real-time PCR to identify the mechanism for

the reduced VEGF protein. There was no significant effect on VEGF gene

expression in any of the treatment groups (1.04 vs. 0.06 relative gene expression

in tumors from saline-treated mice as compared to 1.12 ± 0.12 in tumors from

Ang-(1-7)-treated mice, n = 5 in each group, Figure 21 Panel B). This indicates

that the Ang-(1-7)-mediated reduction In VEGF protein expression is not due to a

reduction in VEGF transcription or a decrease in VEGF mRNA stability.

121

Figure 20: Ang-(1-7) reduces tumor vessel density in triple negative breast

tumors. Panel A contains representative pictures of stained sections of

orthotopic triple negative tumors from mice treated with saline or Ang-(1-7)

following incubation with an antibody to the endothelial cell marker CD34 at a

200x magnification. Quantification of immunoreactive CD34 is depicted in Panel

B and expressed as the average number of CD34 positive cells from three 0.3

mm2 fields at 200x magnification. n = 5, * denotes p < 0.05 relative to treatment

with saline.

122

Panel A

Panel B

Saline Ang-(1-7)

Saline Ang-(1-7)05

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Figure 21: Effect of Ang-(1-7) on VEGF protein and gene expression in

triple negative orthotopic tumors. Panel A. VEGF protein was assessed by

Western blot hybridization and quantified as the density of VEGF

immunoreactivity as a function of EF1α immunoreactivity (n = 5, * denotes p <

0.005 compared to treatment with saline). Panel B. RNA was isolated from

tumor tissue from mice injected with saline or Ang-(1-7) and VEGF mRNA was

quantified by RT real-time PCR. VEGF mean gene expression in saline treated

tumors 1.04 vs. 0.06 vs. 1.12 ± 0.12 VEGF mean relative gene expression in

Ang-(1-7) treated tumors, n=5.

124

Saline Ang-(1-7)0.0

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125

Ang-(1-7) reduces PlGF in triple negative breast tumors

PlGF plays a key role in pathological breast cancer angiogenesis. PlGF was

measured by Western blot hybridization in total protein homogenates isolated

from MDA-MB-231 human breast cancer orthotopic tumors to determine whether

the heptapeptide reduces PlGF in vivo. Ang-(1-7) significantly reduced PlGF

protein in breast orthotopic tumors from mice injected with Ang-(1-7) as

compared to tumors from saline-treated control animals (0.84 ± 0.10 relative

protein expression in tumors from saline-treated mice as compared to 0.35 ±

0.13 in tumors from Ang-(1-7) treated mice, n= 5 in both groups; p < 0.017), as

shown in Figure 22, Panel A. Total RNA was isolated from human breast cancer

orthotopic tumors treated with saline or Ang-(1-7) and PlGF mRNA was

quantified by RT real-time PCR to identify the mechanism for the reduced PlGF

protein. There was no significant effect on PlGF gene expression in any of the

treatment groups (1.04 vs. 0.078 relative gene expression in tumors from saline-

treated mice as compared to 1.00 vs. 0.08 in tumors from Ang-(1-7)-treated mice,

n = 5 in each group, Figure 22, Panel B). This indicates that the Ang-(1-7)-

mediated reduction In PlGF protein expression is not due to a reduction in PlGF

transcription or a decrease in PlGF mRNA stability.

Ang-(1-7) reduces VEGF and PlGF in a time-dependent manner

The reduction of VEGF and PlGF by Ang-(1-7) was also dependent upon

the time of treatment. For these experiments, actively growing MDA-MB-231

cells were treated with 100 nM Ang-(1-7) and harvested at times 0, 1 h, 2 h, and

126

4 h. Protein expression was examined by Western blot hybridization using

specific antibodies to VEGF and PlGF. As observed in Figures 23 and 24, the

reduction in the expression of these two proteins by Ang-(1-7) treatment was

observed after 1 h of treatment with a maximal reduction of 64.72 ± 64.27

percent (n = 6, * denotes p < 0.0002) for VEGF at 4 h and 76.58 ± 6.5 percent at

4 h for PlGF (n = 4, * denotes p < 0.005).

Ang-(1-7) reduces VEGF and PlGF through an AT(1-7) receptor

Actively growing MDA-MB-231 cells were incubated for 4 h in the presence or

absence of Ang-(1-7). As observed in Figure 25 (Panels A and B), treatment

with 100 nM Ang-(1-7) caused a significant reduction in VEGF and PlGF protein

expression (64.3 ± 5.0 percent, n = 5-7, * denotes p < 0.0003 and 60.1 ± 4.5, n =

3-5. * denotes < 0.0036) at 4 h suggesting that Ang-(1-7) reduced VEGF and

PlGF protein expression in triple-negative breast cancer cells in vitro. The

inhibition of VEGF and PlGF in MDA-MB-231 cells by Ang-(1-7) was blocked by

1 µM [D-Pro7]-Ang-(1-7), the selective antagonist for the Ang-(1-7) receptor (4.5 ±

0.32 percent and 20.1 ± 1.2 percent of VEGF and PlGF protein expression,

respectively), as shown in Figure 25 Panels A and B. Treatment with the

receptor antagonist alone had no significant effect and expression was similar to

control levels (17.6 ± 1.3 percent and 19.4 ± 1.3 percent of VEGF and PlGF

protein expression, respectively). This suggests that Ang-(1-7) reduces VEGF

and PlGF in the triple-negative breast cancer cells through the activation of an

AT(1-7) receptor.

127

Figure 22: Effect of Ang-(1-7) on PlGF gene expression in triple negative

breast tumors. Panel A - PlGF protein was assessed by Western blot

hybridization and quantified as the density of PlGF immunoreactivity as a

function of EF1α immunoreactivity and expressed as a percentage of the Control

(n = 5, * denotes p < 0.005 compared to saline treatment). Panel B - RNA was

isolated from tumor tissue of mice injected with saline or Ang-(1-7) and PlGF

mRNA was quantified by RT real-time PCR. PlGF gene expression in saline

treated tumors was 1.034 ± 0.08 vs. 1.00 ± 0.08 PlGF mean relative gene

expression in Ang-(1-7) treated tumors, n = 5.

128

Saline Ang-(1-7)0.0

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Panel A

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129

Figure 23: Ang-(1-7) reduces VEGF protein expression in a time-dependent

manner. Effect of Ang-(1-7) VEGF was determined Western blot hybridization.

The data are presented as relative VEGF immunoreactivity using EF1α as a

loading control. MDA-MB-231 breast cancer cells treated with 100 nM Ang-(1-7)

for 4 h. n = 3 – 6, * denotes p < 0.005 relative to control at time zero.

130

0 1 2 3 40

25

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125

* **

Time (hours)

VEG

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131

Figure 24: Ang-(1-7) reduces PlGF protein expression in a time-dependent

manner. Effect of Ang-(1-7) PlGF was determined Western blot hybridization.

The data are presented as relative PlGF immunoreactivity as protein using EF1α

as a loading control. MDA-MB-231 breast cancer cells were treated with 100 nM

Ang-(1-7) for 4 h. n = 3 – 6, * denotes p< 0.005 relative to time zero.

132

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40

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133

Figure 25: Ang-(1-7) reduces VEGF and PlGF through an AT(1-7) receptor.

Protein expression was analyzed by Western blot hybridization using antibodies

against VEGF and PlGF; an EF1α antibody was used as a loading control. Cells

were incubated for 4 h with no addition (Control), 100 nM Ang-(1-7), [D-Pro7]-

Ang-(1-7) alone (DP), or Ang-(1-7) in the presence of 1 µM [D-Pro7]-Ang-(1-7)

[DP + A7]. n = 4, * denotes p < 0.05 relative to control.

134

Control

Ang-(1-7) DP

DP+A7

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Control

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DP+A7

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135

Ang-(1-7) reduces VEGF and PlGF through an up-regulation in DUSP1

The expression of the VEGF family of cytokines is controlled in part

through the activities of MAP kinases (154). We observed that Ang-(1-7)

reduces MAP kinase activity in breast cancer cells and this reduction was

associated with an up-regulation of DUSP1 mRNA and protein (Figures 26,

Panel A and B). This suggests that a decrease in DUSP1 would reverse the

Ang-(1-7)-mediated reduction in VEGF and PlGF in breast cancer cells. siRNA’s

against DUSP1 and cyclophilin were transfected into actively growing MDA-MB-

231 cells. Cyclophilin served as a control since it has no known homology to

gene sequences in humans. After a 48 h incubation, breast cancer cells were

treated with 100 nM Ang-(1-7); a group of cells were transfected but left

untreated to serve as a control. As shown in Figures 32, A and B, Ang-(1-7)

caused a 56.8% and 50.9% reduction in VEGF and PlGF, respectively, in cells

transfected with cyclophillin siRNAs. The reduction was comparable to the levels

observed in non-transfected control cells. In contrast, treatment with Ang-(1-7) in

cells transfected with siRNAs to DUSP1 showed no reduction in VEGF or PlGF

which were comparable to the cyclophillin control group. Treatment with siRNAs

to DUSP1 alone showed no significant effect in the expression of VEGF and

PlGF. A 62.1% reduction in DUSP1 was observed following incubation with the

DUSP1-specific siRNAs (Figure 27). Taken together, these results suggest that

the Ang-(1-7)-mediated decrease in VEGF and PlGF is due to the up-regulation

of DUSP1 and a consequential reduction in MAP kinase activity.

136

Figure 26: Ang-(1-7) reduces VEGF and PlGF through an up-regulation in

DUSP1. Semi-confluent MDA-MB-231 cells were transfected with either DUSP1

or cyclophilin (Cyclo) siRNAs using Oligofectamine. After 48 h, transfected cells

were incubated for an additional 4 h with or without 100 nM Ang-(1-7) [A7].

Protein expression was analyzed by Western blot hybridization using antibodies

against VEGF and PlGF; an EF1 alpha antibody was used as a loading control.

n = 5 – 6, * denotes p < 0.005 relative to cycophilin.

137

Cyclophilin

Cyclo +

A7

DUSP1

DUSP1 + A

70

25

50

75

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125

*

VEG

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Panel A

Cyclophilin

Cyclo +

A7

DUSP1

DUSP1 + A

70

25

50

75

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125

150

175

*PlG

F Im

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lGF/

EF1

α)

Panel B

VEGF

EF1α

Cyc

loph

illin

Cyc

loph

illin

+ A

7

siR

NA

DU

SP1

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SP1

+ A

7

VEGF

EF1α

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loph

illin

Cyc

loph

illin

+ A

7

siR

NA

DU

SP1

siR

NA

DU

SP1

+ A

7

138

Figure 27: Gene knockdown with siRNAs to DUSP1 in MDA-MB-231 cells.

To determine the reduction in DUSP1 by DUSP1 siRNA, DUSP1 was measured

by Western blot hybridization using specific antibodies to DUSP1. Treatment

with DUSP1 siRNAs caused a 62.1% reduction in DUSP1 expression. n = 4; *

denotes p < 0.02 relative to cells transfected with cyclophilin.

139

Cyclophilin

siRNA

DUSP1 siR

NA0

20

40

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100

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USP

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140

Inhibition of endothelial cell tubule formation by Ang-(1-7)

The effect of Ang-(1-7) on the formation of tubules in Matrigel was determined to

demonstrate a direct effect of the heptapeptide on endothelial cells. Human

endothelial cells of the EA.hy926 cell line were plated at a density of 1 x 104

cells/mL into individual wells of a 96-well cluster dish coated with Matrigel, in the

presence or absence of Ang-(1-7). Untreated endothelial cells formed a network

of multiple tube-like structures, as seen in Panel A of Figure 28, while the number

of tube-like structures was significantly decreased in the presence of 100 nM

Ang-(1-7) [38.7 ± 5.1 in the Control group compared to 17.8 ± 3.2 in the Ang-(1-

7)-treated group; n = 3; p < 0.05] (Figure 28, Panel B). Endothelial cells also

were incubated with 100 nM Ang-(1-7) in the presence of 1 µM Ang-(1-7)

receptor antagonist [D-Pro7]-Ang-(1-7), to determine whether the inhibition by the

heptapeptide was a receptor-mediated process. As shown in Figure 28, the

number of branch points in the presence of Ang-(1-7) and the AT(1-7) receptor

antagonist [D-Pro7]-Ang-(1-7) did not differ from the Control, indicating that the

inhibition of endothelial tubule formation required activation of an AT(1-7) receptor.

The antagonist alone had no effect on tubule formation. Both mas mRNA and

protein were detected in these cells by RT real-time PCR suggesting that mas

may be involved in the process.

Endothelial cells (104) were seeded onto Matrigel in the absence or

presence of increasing concentrations of Ang-(1-7) [from 0.5 to 100 nM] to

determine whether the inhibition of tubule formation was dependent on the dose

141

of the heptapeptide. After 16 h, the number of branch points was counted and

compared to the Control group, in the absence of the Ang-(1-7). As shown in

Figure 29, Ang-(1-7) reduced the number of branch points in a dose-dependent

manner, with a maximal reduction greater than 50% at a 10 nM concentration.

HUVECs (104 cells) were seeded onto Matrigel, in the absence or

presence of 10 nM Ang-(1-7), to confirm the reduction in tubule formation in a

non-transformed cell line. As shown in Figure 30, treatment with Ang-(1-7)

caused an approximate 50% reduction in HUVEC tubule formation as compared

to control cultures (26.0 ± 7.0 vs. 52.8 ± 7.0, n = 3, p < 0.05 ). HUVECs also

were incubated with 10 nM Ang-(1-7) in the presence of 100 nM Ang-(1-7)

receptor antagonist [D-Pro7]-Ang-(1-7) to determine whether the inhibition by the

heptapeptide was a receptor-mediated process. Branch point number in the

presence of Ang-(1-7) and [D-Pro7]-Ang-(1-7) did not differ from the Control (52.8

± 12.2 vs. 44.3 ± 12.2), while the AT(1-7) receptor antagonist alone had no effect

on tubule formation (52.8 vs. 48.0 ± 2.8). This experiment demonstrates that

Ang-(1-7) effectively inhibits HUVEC tubule formation by activating an AT(1-7)

receptor.

142

Figure 28: Ang-(1-7) inhibition of endothelial cell tubule formation.

EA.hy926 cells were seeded onto Matrigel alone (Control) or with 10 nM Ang-(1-

7), with 10 nM Ang-(1-7) and 100 nM AT(1-7) receptor antagonist [D-Pro7]-Ang-(1-

7) (D-Pro), or with 100 nM [D-Pro7]-Ang-(1-7) alone. After 16 h, the cells were

photographed to visualize and quantify tubule formation. A representative

photograph of each treatment is shown in Panel A and the quantification of

branch points is depicted in Panel B. n = 3, * denotes p < 0.05 relative to control.

143

Panel A

Panel B

Control Ang-(1-7) Ang-(1-7) D-Pro0

5

10

15

20

25

30

35

40

45

*

+ D-Pro

Num

ber o

f Tub

ules

144

Figure 29: Dose-dependent reduction of tubule formation by Ang-(1-7).

EA.hy926 cells were seeded onto Matrigel alone (Control) or with increasing

concentrations of Ang-(1-7). After 16 h, the cells were photographed to visualize

tubule formation and branch points were quantified. n = 3 – 5, * denotes p < 0.01

compared to treatment at time zero.

145

0 0.5 1 10 50 1000

20

40

60

80

100

** * *

Ang-(1-7) [nM]

Num

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)

146

Figure 30: Ang-(1-7) inhibition of HUVEC tubule formation. Cells were

seeded onto Matrigel alone (Control) or with 10 nM Ang-(1-7) (A7) or with 10 nM

Ang-(1-7) [A7] and 100 nM AT(1-7) receptor antagonist [D-Pro7]-Ang-(1-7) (DP), or

with 100 nM [D-Pro7]-Ang-(1-7) alone. After 16 h, the cells were tubule formation

was quantified under a light microscope. * p < 0.05, n = 3 relative to control.

147

Control A7 DP A7 + DP0

20

40

60

80

*

Num

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148

Ang-(1-7) Inhibits endothelial cell proliferation

Studies in our laboratories showed that Ang-(1-7) inhibits proliferation by

reducing mitogen-stimulated DNA synthesis and cell growth in VSMCs (30) and

in lung cancer cells (60). The reduction in serum-stimulated DNA synthesis by

Ang-(1-7) in both cell types was dose-dependent with EC50s in the sub-

nanomolar range. EA.hy926 endothelial cells were stimulated with 1% FBS and

treated with increasing concentrations of Ang-(1-7) (10-12-10-6 M) to determine

whether the heptapeptide reduces endothelial cell growth. After 48 h, 3H-

thymidine incorporation was measured as an indication of cell proliferation. Ang-

(1-7) attenuated EA.hy926 cell growth with a maximal effect dose at 1 μM.

Maximal inhibition by 1 μM Ang-(1-7) was approximately 50% of control (Figure

31). Thus, Ang-(1-7) inhibits mitogen-stimulated DNA synthesis, suggesting that

Ang-(1-7) inhibits endothelial cell proliferation during angiogenesis.

Ang-(1-7) reduction of angiogenesis in the Chick Chorioallantoic Membrane (CAM)

The anti-angiogenic effect of Ang-(1-7) was determined in vivo by

measuring neovascularization during the development of the chick embryo.

Tyson Farm eggs were incubated at 37°C for four days. At day four, the air cell

was removed and on day 6, DMEM media containing 1% albumin with or without

100 nM Ang-(1-7) was added. Development was observed 24 h after treatment

(day 7). Vessel formation was stimulated in the presence of media enriched with

1% albumin (Figure 32, left panel); on the other hand, treatment of chicken

149

Figure 31: Effect of Ang-(1-7) endothelial cell proliferation. EA.hy926 cells

were treated with increasing Ang-(1-7) concentrations and 3H-thymidine was

measured as an indication of DNA synthesis. n = 3.

150

-10 -9 -8 -70

25

50

75

100

Ang-(1-7) [log M]

3 H-T

hym

idin

e In

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(Per

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151

embryos with Ang-(1-7) in the media caused a significant reduction in branch

point formation (Figure 32) (58.6 ± 3.6 branch points vs. 29.5 ± 3.6, n = 3 – 4, p <

0.05). This indicates that treatment with Ang-(1-7) participates in the inhibition of

angiogenesis in an in vivo model.

In another set of experiments, methylcellulose discs containing saline or

100 nM of Ang-(1-7) were placed on the surface of the CAM in an avascular area

to specifically target an area of active embryonic vascularization. After 24 h of

treatment, the discs were removed and the number of branch points was

quantified. As shown in Panel A of Figure 33, vessels continued to develop in

the saline-treated Control group, while vessel formation was significantly reduced

in embryos treated with Ang-(1-7) [21.1 ± 2.0 branch points in the Control group

compared to 9.3 ± 1.1 branch points in the Ang-(1-7)-treated group; n = 9; p <

0.001]. In an additional group of chick embryos, Ang-(1-7) was added in the

presence of the receptor blocker [D-Pro7]-Ang-(1-7) to determine whether the

inhibition of vessel formation was due to receptor activation. As shown in Figure

33, the addition of the receptor antagonist completely blocked the response to

Ang-(1-7), indicating that the reduction in neoangiogenesis was mediated by an

AT(1-7) receptor. Incubation with 1 uM [D-Pro7]-Ang-(1-7) alone had no effect on

vessel formation in the CAM.

152

Figure 32: Ang-(1-7) inhibits angiogenesis in vivo. In vivo effects of Ang-(1-

7) in the chicken embryo (left panel). Eggs were incubated for 4 days and were

treated with media containing albumin (Control) with or without Ang-(1-7).

Neovascularization was quantified at day 6 by comparing pictures taking at the

start and at the end of the treatment (n = 5, * denotes p < 0.05 relative to control).

153

Control Ang-(1-7) Control Ang-(1-7)0

10

20

30

40

50

60

*

Day 4 Day 6B

ranc

h Po

ints

Control Control

Ang-(1-7)Ang-(1-7)

154

Figure 33: Inhibition of neovascularization in the CAM by Ang-(1-7) is

mediated by an AT(1-7) receptor. Methylcellulose disks containing saline, 100

nM Ang-(1-7), 100 nM Ang-(1-7) and 1 µM AT(1-7) receptor antagonist [D-Pro7]-

Ang-(1-7) (D-Pro), or 1 µM [D-Pro7]-Ang-(1-7) alone were placed in an avascular

area of the CAM. After 24 h, the disks were photographed and branch points

were quantified to assess vessel formation. A representative photograph of each

treatment is shown in Panel A and the quantification of branch points is depicted

in Panel B. n = 9 – 12, * denotes p < 0.001 relative to control.

155

Control Ang-(1-7) Ang-(1-7) D-Pro0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

+ D-Pro

*

Bra

nch

Poin

ts/7

cm

Are

a

Panel A Panel B

156

Subcutaneous injection of Ang-(1-7) inhibits lung tumor volume

Athymic mice bearing a human A549 lung tumor were administered

subcutaneous injections of either saline or Ang-(1-7) at a concentration of 1000

µg/kg/day. The mice were injected daily, for 5 days, followed by a 2 day rest

period, and the injections were continued for 6 weeks. As shown in Panel A of

Figure 34, tumor volume in the two treatment groups was similar at the initiation

of treatment [108.8 ± 3.9 mm3 in the saline-treated group compared to 110.5 ±

3.1 mm3 in the Ang-(1-7)-treated group; n = 5 in each group]. The tumors of

saline-treated mice increased in size over time, while the growth of tumors from

mice treated with Ang-(1-7) was reduced markedly by day 42 [752.5 ± 46.9 mm3

in the saline-treated group compared to 265.5 ± 27.4 mm3 in the Ang-(1-7)-

treated group; n = 5 in each group; p < 0.0001]. The animals maintained their

body weight as well as food and water consumption irrespective of treatment and

showed no evidence of reduced motor function.

At the end of the study, the mice were euthanized and the tumors were

removed and weighed. As shown in Panel B of Figure 34, the tumors from mice

treated with the heptapeptide weighed almost 60% less than the tumors of mice

infused with saline [0.72 ± 0.06 g in the Ang-(1-7)-treated group versus 1.66 ±

0.12 g in the saline-treated group; n = 5 in each group; p < 0.005]. No gross

pathologic abnormalities were observed in major organs following sacrifice,

demonstrating a lack of toxic side effects at the Ang-(1-7) dose given.

157

Figure 34: Effect of Ang-(1-7) on human lung cancer xenograft growth.

Panel A - The size of human A549 lung tumor xenografts from mice injected with

saline or 1000 µg/kg/day was measured using a caliper and volume was

calculated using the formula for a semi-ellipsoid (4/3πr3)/2). n = 5, * denotes p <

0.05 compared to treatment with saline. Panel B - The tumors from mice infused

with either saline or Ang-(1-7) were weighed at the time of sacrifice. n = 5, *

denotes p < 0.005 relative to treatment with saline.

158

0 5 10 15 20 25 30 35 40 450

100

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300

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500

600

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800

900SalineAng-(1-7)

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** *

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Tum

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Panel A

Saline Ang-(1-7)0.0

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ght (

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159

Ang-(1-7) Reduces Angiogenesis in Human Lung Tumor Xenografts

Subcutaneous lung tumors were immunostained for CD34, an endothelial

cell marker, to determine whether the reduction in lung tumor growth observed

with Ang-(1-7) treatment was associated with a decrease in tumor blood vessel

formation. Blood vessels were located throughout the tumor; clusters of vessels

or vessel “hot spots” were localized mainly in the periphery of the tumors.

Tumors from animals administered saline or Ang-(1-7) showed a similar quantity

of hotspots; however, the number of vessels in the clusters from mice injected

with the heptapeptide was reduced significantly. Tumor tissue sections from

saline-treated animals showed strong immunoreactivity to CD34 when compared

to sections from Ang-(1-7)-treated animals (Figure 35, Panel A). As shown in

Panel B of Figure 35, injection of Ang-(1-7) caused an approximate 50%

reduction in tumor vessel density (average of 6 fields) when compared to

sections from saline-treated animals [25.1 ± 4.9 immunopositive CD34 cells/field

in the saline-treated group compared to 10.6 ± 2.1 in the Ang-(1-7)-treated group;

n = 5 in each group; p < 0.03], suggesting that the heptapeptide reduces

angiogenesis to inhibit lung tumor growth

Ang-(1-7) reduces VEGF in human lung tumor xenografts

VEGF is the primary pro-angiogenic factor, released from cancer cells to

stimulate the growth of new blood vessels from pre-existing vessels. VEGF was

measured by Western blot hybridization in total protein homogenates isolated

from A549 human lung tumor xenografts to determine whether the heptapeptide

160

reduces VEGF in vivo. Ang-(1-7) significantly reduced VEGF protein in tumor

xenografts from mice injected with Ang-(1-7) as compared to tumors from saline-

treated control animals [1.08 ± 0.18 relative protein expression in tumors from

saline-treated mice as compared to 0.15 ± 0.08 in tumors from Ang-(1-7) treated

mice, n = 5 in each group; p < 0.002], as shown in Figure 36A.

Total RNA was isolated from human lung tumor xenografts treated with

saline or Ang-(1-7) and VEGF mRNA was quantified by RT real-time PCR to

identify the mechanism for the reduced VEGF protein. A greater than 50%

decrease in VEGF mRNA was observed in the human lung tumor xenografts

from mice administered heptapeptide as compared to controls [1.01 ± 0.10

relative gene expression in tumors from saline-treated mice as compared to 0.43

± 0.06 in tumors from Ang-(1-7)-treated mice, n = 5 in each group; p < 0.001]

(Figure 36, Panel B), suggesting that the Ang-(1-7)-mediated reduction in VEGF

was due to either a transcriptional regulatory mechanism or a decrease in VEGF

mRNA stability.

Similarly, the parent human A549 lung cancer cells were incubated with

Ang-(1-7) to assess the time-dependent inhibition of VEGF by the heptapeptide.

Actively-growing A549 cells were treated with 100 nM Ang-(1-7) for various

periods of time between 4 and 24 h and VEGF was quantified by Western blot

hybridization. Treatment of the human lung cancer cells with the heptapeptide

caused a marked decrease in VEGF protein, with a maximal reduction of 55.5 ±

161

12.8% after a 12 h incubation with Ang-(1-7) (Figure 37, Panel A). Administration

of 100 nM Ang-(1-7) also caused a time-dependent decrease in VEGF mRNA in

human A549 cells between 1 to 8 h of treatment (Figure 37, Panel B), with a

maximal reduction at 2 and 4 h (46.2 ± 2.8 and 41.0 ± 2.4 percent reduction in

VEGF gene expression respectively). In another set of experiments A549 cells

where incubated for 4 h with 100 nM Ang-(1-7) which caused a 51.8 ± 4.1

percent reduction in VEGF mRNA expression which is comparable to the

reduction observed at 4 h in the time course experiments. Treatment with the

Ang-(1-7) receptor antagonists [D-Pro7]-Ang-(1-7) or [D-Ala7]-Ang-(1-7)

completely blocked the Ang-(1-7)-mediated decrease in VEGF mRNA (-0.066 ±

0.54 and -0.090 ± 0.72 mean difference of gene expression from control,

respectively) while the antagonists alone had no effect (Figure 37, Panel C),

indicating that the heptapeptide activated an AT(1-7) receptor to reduce VEGF.

These in vitro results provide further support that Ang-(1-7) may selectively

decrease VEGF to reduce angiogenesis.

162

Figure 35: Inhibition of human lung tumor angiogenesis by Ang-(1-7).

Panel A contains representative pictures of stained sections of tumors from mice

treated with saline or Ang-(1-7) following incubation with an antibody to the

endothelial cell marker CD34 at a 200x magnification. Quantification of

immunoreactive CD34 is depicted in Panel B and expressed as the average

number of CD34 positive cells from six 0.3 mm2 fields at 200x magnification. n =

5, * denotes p < 0.03 relative to treatment with saline.

163

Panel A Panel B

Saline Ang-(1-7)0

5

10

15

20

25

30

*C

D34

Imm

unor

eact

ivity

(CD

34 p

ositi

ve v

esse

ls/fi

eld)

164

Figure 36: Reduction of VEGF in human A549 lung tumor xenografts by

Ang-(1-7). Panel A - VEGF protein was assessed by Western blot hybridization

and quantified as the density of VEGF immunoreactivity as a function of α-actin

immunoreactivity in tumor tissue from human lung cancer xenografts treated with

saline or Ang-(1-7). n = 5, * denotes p < 0.002 compared to treatment with

saline. Panel B - RNA was isolated from tumor tissue from mice injected with

saline or Ang-(1-7) and VEGF mRNA was quantified by RT real-time PCR. n = 5,

* denotes p < 0.001 compared to treatment with saline.

165

Saline Ang-(1-7)0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*VEG

F Im

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ctiv

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EGF/β -

Act

in)

Panel A

Saline Ang-(1-7)0.0

0.2

0.4

0.6

0.8

1.0

1.2

*VEG

F m

RN

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Panel B

166

Figure 37: The reduction of VEGF by Ang-(1-7) is time-dependent and

mediated by an AT(1-7) receptor. A549 cells were incubated with 100 nM Ang-

(1-7) from 4 h – 24 h. VEGF protein was assessed by Western blot hybridization

and quantified as the density of VEGF immunoreactivity as a function of α-actin

immunoreactivity and expressed as a percentage of the Control at time zero, in

Panel A. RNA was isolated from tumor tissue from mice injected with saline or

Ang-(1-7) and VEGF mRNA was quantified by RT real-time PCR, in Panel B. In

Panel C, cells were incubated for 4 h with no addition (Con), 100 nM Ang-(1-7)

[A7], Ang-(1-7) in the presence of 1 µM [D-Pro7]-Ang-(1-7) (DP), [D-Pro7]-Ang-(1-

7) alone, Ang-(1-7) in the presence of 1 µ M [D-Ala7]-Ang-(1-7) (DA), or [D-Ala7]-

Ang-(1-7) alone. n = 4, * denotes p < 0.05 compared to time zero (Panels A and

B) or Control (Panel C).

167

0 4 8 12 16 20 240

20

40

60

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100

**

*

Time (hours)

VEG

F Im

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)

0 2 4 6 80.0

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Panel A Panel B

Con A7A7/D

P DPA7/D

A DA0.0

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Panel C

168

CHAPTER IV DISCUSSION

Studies from our laboratory were the first to characterize the anti-growth

properties of Ang-(1-7) on actively proliferating vascular cells (20;30;35;60). This

led to the investigation of the heptapeptide as an anti-mitogenic agent for cancer.

We reported that the Ang-(1-7)-mediated reduction in lung cancer cell growth in

vitro was mediated by the activation of a unique AT(1-7) receptor and a decrease

in MAP kinase activities (60). In addition, Ang-(1-7) reduced the in vivo growth of

lung tumor xenografts in athymic mice; the observed reduction in tumor growth

was associated with a significant decrease in cell proliferation and a reduction in

COX-2 (146). Ang-(1-7) not only arrested the growth of lung tumors but also

caused a more than 30% reduction in tumor size, suggesting that the

heptapeptide activated apoptosis or reduced angiogenesis. The purpose of

these studies was to investigate the mechanisms underlying the reduction of

tumor growth by Ang-(1-7) and to extend the investigation to breast cancer cells,

specifically the triple negative subtype, to determine whether the anti-tumorigenic

effects of Ang-(1-7) include a reduction in tumor angiogenesis.

Mechanisms of Ang-(1-7) Inhibition of Triple Negative Breast Orthotopic Tumors

Our studies illustrate for the first time the effectiveness of Ang-(1-7) in

controlling in vivo human breast cancer growth in an orthotopic mouse model.

MDA-MB-231 cells, injected into the mammary fat pad of athymic mice, form

tumors that are characteristic of basal types of breast cancer and fall into the

169

category of “triple negative” since they do not express the estrogen receptor or

the progesterone receptor and do not over-express HER2 (155). Tumors from

animals treated with Ang-(1-7) showed a significant decrease in volume when

compared to tumors of animals treated with saline. The mean tumor volumes of

the two treatment groups were comparable prior to the administration of either

saline or Ang-(1-7); however, the mean tumor volume of saline-treated animals

was about 4-fold higher at the end of the 30 day treatment. This indicates that

the subcutaneous administration of Ang-(1-7) prevented the growth of breast

orthotopic tumors in an in vivo athymic mouse model. This corroborates the anti-

tumor actions of Ang-(1-7) previously reported in vitro in lung cancer cells and in

vivo in a lung tumor xenograft model (60;146).

Injection of MDA-MB-231 breast cancer cells into the mammary fat pad of

athymic mice investigates the efficacy of Ang-(1-7) for reducing breast tumor

growth at a clinically relevant site, since the tumors grow in a microenvironment

similar to the primary site of development. It was previously demonstrated that

agents exhibiting anti-cancer properties in orthotopic models of cancer were

more effective in clinical trials (156). Using an in vivo orthotopic model of breast

cancer also increases the chances of metastasis. Although we did not observe

any metastasis to distant organs from the primary site in our studies, the 30 day

treatment period used in this study may have been too short, as metastasis is a

late stage event in carcinogenesis.

170

Ang-(1-7) was administered to mice by subcutaneous injections at a dose

of 1000 μg/kg every 12 hours for 30 days. This concentration was selected

based on dose escalation studies in nude mice performed in our laboratory

(Gallagher and Tallant, unpublished results). Pathological assessment after

sacrifice showed no toxic side effects or anatomical abnormalities after treatment

with Ang-(1-7). This lack of toxicity is in agreement with observations from our

previous studies in which Ang-(1-7) was infused into rodents at a dose of 24

μg/kg/h, using osmotic mini-pumps (20;146). Others also infused this dose of

Ang-(1-7) into animals without observing changes in body weight, heart rate or

blood pressure (22). The lack of toxic side effects observed in our study is also

in agreement with a previous report that showed no adverse side effects when

the heptapeptide was administered to patients as adjuvant therapy for cytopenia

during chemotherapy (23).

Previously, we showed that the reduction in human lung tumor xenograft

growth with Ang-(1-7) treatment was linked to a decrease in COX-2. COX-2

activates signaling pathways essential in the progression of mammary

carcinogenesis, such as proliferation, angiogenesis and metastasis (151;157).

Studies suggest that inhibition of COX-2 by the use of NSAID’s reduced the

overall risk for development of breast cancer (relative risk [RR] = 0.88, 95%

confidence interval [CI] = 0.84 to 0.93) (158;159). The expression of COX-2 in

breast cancer is not dependent on hormonal receptor status and COX-2 is highly

over-expressed in HER2-amplified breast cancer (159). Based on these reports,

171

we expected that COX-2 would be expressed in our MDA-MB-231 breast tumor

samples and this was confirmed by RT real-time PCR analysis. However,

contrary to the molecular mechanism of the growth reduction reported in our

studies with human lung tumor xenografts, we observed no significant effect on

COX-2 gene expression with Ang-(1-7) treatment in our experiments with human

breast orthotopic tumors. Studies from our laboratory suggest that Ang-(1-7)

may mediate anti-growth effects by shifting the balance between pro-mitogenic

and ant-mitogenic prostaglandins (Gallagher and Tallant, unpublished results).

However, Ang-(1-7) administration did not alter PGES gene expression in the

triple negative breast tumors, suggesting that the inhibition observed by the

heptapeptide was not mediated by reduced COX-2 activity or the downstream

growth promoting prostaglandins.

We previously speculated that the reduction in proliferation observed with

Ang-(1-7) treatment in lung tumor xenografts could be due to the arrest of cell

cycle progression or through the inhibition of signaling pathways that regulate cell

survival (146) The marked difference in growth rate and volume observed during

the development of orthotopic breast tumors treated with either saline or Ang-(1-

7) was apparent following immunohistochemical analysis of tumor sections using

the proliferation marker Ki67. Tumors of animals administered saline

demonstrated strong immunoreactivity against Ki67 as compared to the low

immunoreactivity of Ang-(1-7)-treated tumors. This indicates that treatment with

Ang-(1-7) may negatively control progression of the cell cycle or inhibit signaling

172

cascades that stimulate cell proliferation. The reduced Ki67 immunostaining of

Ang-(1-7)-treated tumors corroborates our previous studies in lung tumor

xenografts as well as the anti-proliferative effects of Ang-(1-7) observed by us in

vascular studies (20;30;35;39;60).

We demonstrated that Ang-(1-7) significantly reduced MAP kinase activity

in triple negative breast tumors. The regulation of MAP kinase activities is

extensively characterized in cancer cells and it is known that MAP kinases,

especially ERK1 and ERK2, are up-regulated in breast cancer (160-162). The

observed inhibition of ERK1 and ERK2 activity by Ang-(1-7) is in agreement with

previous studies demonstrating that the anti-growth effect of Ang-(1-7) is

associated with a reduction in MAP kinase activity in actively proliferating

vascular cells (30;60;163;164).

In cancer the sequential phosphorylation of MAP kinase kinase kinases

(MAPKKKs or Raf), MAP kinase kinases (MAPKKs or MEKs) and MAP kinases

(such as ERK1 and ERK2) leads to the phosphorylation of substrates that are

involved in the in the regulation of various cellular process that results in cell

growth and survival. MAP kinases, specifically ERK1 and 2, are negatively

regulated by dual specificity phosphatases (DUSPs), also known as MAP kinase

phosphatases (MKPs), which are capable of removing both phosphotyrosine and

phosphothreonine from their protein targets (165). Since we previously showed

that Ang-(1-7) inhibits mitogen-stimulated cell proliferation, Ang-(1-7) could

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reduce the production of growth factors to inhibit the receptor-mediated activation

of MAP kinase activities. Ang-(1-7) could also inhibit the activity of upstream

MAP kinase kinases such as MEK1/2 to decrease ERK activation. Alternatively,

the heptapeptide could activate or up-regulate a dual specificity phosphatase to

deactivate ERKs and inhibit their activity. Previous studies by our group showed

that the Ang-(1-7)-mediated inhibition of VSMC growth was prevented by pre-

treatment with phosphatase inhibitors, suggesting that Ang-(1-7) may activate or

up-regulate a MAP kinase phosphatase to inhibit cell growth (30).

We determined by RT real-time PCR and Western blot hybridization that

the DUSP1 gene and protein are increased in MDA-MB-231 tumors from animals

treated with Ang-(1-7) when compared to tumors from animals administered

saline. In cultured MDA-MB-231 cells, we demonstrated a similar sustained

expression of DUSP1 following incubation with Ang-(1-7). Treatment of cells with

Ang-(1-7) in combination with its receptor antagonists, [D-Ala7]-(Ang-(1-7) and

[D-Pro7]-(Ang-(1-7), showed that blockade of the AT(1-7) receptor prevented the

Ang-(1-7)-mediated up-regulation DUSP1, suggesting that the effect of the

heptapeptide on DUSP1 is mediated by an AT(1-7) receptor. These data

demonstrate that the observed inhibition of the MAP kinases ERK1 and ERK2 is

mediated by the AT(1-7) receptor-activated up-regulation of DUSP1 and the

associated increase in phosphatase activity to dephosphorylate and deactivate

ERK1 and ERK2.

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DUSP, also known as MKP-1, is an inducible nuclear protein that is

implicated in cell cycle control (141) and is regulated at transcription in response

to factors that stimulate ERK MAP kinases (142). Recent reports demonstrated

that DUSP1 is also regulated by ERK1/ERK2 through a negative feedback

mechanism to reduce phosphatase activity by polyubiquitination of the DUSP1

protein, targeting it for proteosomal degradation (143). Since DUSP1

dephosphorylates threonine and tyrosine residues to deactivate MAP kinases,

increased expression of DUSP1 may lead to decreased ERK activity and a

reduction in cell proliferation and survival. Over-expression of DUSP1/MKP-1 in

VSMCs resulted in inactivation of MAP kinases, including ERK1/ERK2 and

inhibition of DNA synthesis (144). In a another study, treatment with sildenafil

inhibited proliferation of pulmonary smooth muscle cell; the inhibition in

proliferation was associated with a reduction in MAP kinase activity and an up-

regulation of the DUSP1/MKP-1 mediated through cyclic GMP or cyclic GMP-

dependent kinase I alpha. In the same study, exposure to sodium vanadate, a

tyrosine phosphatase inhibitor, prior to treatment with sildenafil reversed the

inhibition of ERK phosphorylation and prevented the anti-proliferative effects

observed by sildenafil treatment (166). These findings are in agreement with

observations from our laboratory in which treatment with phosphatase inhibitors

abolished the anti-growth effect of Ang-(1-7) in VSMCs (30;32).

DUSP1/MKP-1 is highly expressed in vascular tissue (167). However,

MKP1/DUSP1 is down-regulated in rapidly proliferating VSMCs in the neointima

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following balloon injury of the rat carotid artery, in association with an increase in

the activities of ERK1 and ERK2 (168). In a similar model of injury to the rat

carotid artery, treatment with Ang-(1-7) prevented VSMC growth to reduce

neointimal formation (41), Over-expression of MKP-1 in arterial smooth muscle

cells caused growth arrest in the G1 phase and entry into the S phase of the cell

cycle (168). These results indicate that up-regulation of DUSP1 negatively

regulates ERK1/ERK2 and cell cycle promoting proteins, leading to cell cycle

arrest and a reduction in cell proliferation. This suggests that agents which up-

regulate DUSP1/MKP-1 may be useful in the treatment of diseases in which MAP

kinases are up-regulated and cells are rapidly proliferating, such as in breast

cancer.

Activated MAP kinases are highly expressed in breast cancer and have a

critical role in the progression of breast tumors, independent of hormonal status

and amplification of HER2 (162). Results from Umemura et al. suggest that

ERK1ERK2 is more highly expressed in triple negative breast cancer when

compared with other subtypes of breast cancer (82). Agents which up-regulate

DUSP1/MKP-1 were used in the treatment of breast cancer (137;153). Breast

cancer cells were treated with nitric oxide (NO) donors to induce apoptosis,

which was associated with inhibition of both ERK1/ERK2 and Akt activities; NO

was unable to deactivate ERK1/ERK2 or Akt or induce apoptosis in the presence

of a phosphatase inhibitor, suggesting that inactivation of these kinases and

stimulation of apoptosis is dependent on phosphatase activity (153) The

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Bowman-Birk inhibitor (BBI) is a protease inhibitor which has anti-cancer

properties and is currently in a human phase II clinical trial (169). The molecular

mechanisms of BBI inhibition of carcinogenesis were studied in breast cancer

cells and included a reduction in proteins that promote progression of the cell

cycle and down-regulation of ERK1/ERK2 (169); dephosphorylation and

deactivation of ERK1/ERK2 was blunted by treatment with phosphatase

inhibitors. Subsequent experiments showed that BBI up-regulated DUSP1/MKP-

1 in breast cancer cells, suggesting that the anti-proliferative effects of BBI are

mediated, in part, through direct up-regulation of DUSP1/MKP-1.(169). These

data are in agreement with our results showing that Ang-(1-7) inhibits the growth

of triple-negative breast tumors through a receptor-mediated up-regulation of

DUSP1/MKP-1. Collectively, these results suggest that agents which up-regulate

DUSP1/MKP-1, such as Ang-(1-7), should be considered in the treatment of

breast cancers.

There is an association between the over-expression of MKP-1 and

chemoresistance of breast cancer following treatment with alkylating agents,

anthracyclines, and microtubule inhibitors (170). Related studies suggested that

MKP-1 is over-expressed in human breast tumors, associating its expression

with progression of carcinogenesis (171). Over-expression of DUSP-1/MKP-1

could result from the elevated expression and activity of ERK1/ERK2 in these

breast tumors, since up-regulation of MAP kinases is associated with an increase

in DUSP1/MKP-1 activity (137). Although MKP-1 dephosphorylates and

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inactivates ERK1/ERK2, it also deactivates the MAP kinases JNK and p38

(137;172). Since JNK/p38 kinases are implicated in the induction of apoptosis

(170), a reduction in their activities due to increased MKP-1 activity could be

associated with chemoresistance in breast tumors (170). This awaits further

investigation. However, if an increase in MKP-1 is associated with

chemoresistance to certain cytotoxic agents, combination treatments of Ang-(1-7)

with these cytotoxic therapies may result in an increase in the efficacy of the

cytotoxic agents. Further studies will need to address the use of Ang-(1-7) in

combination with cytotoxic agents to assess the combinatorial effect on tumor

growth.

The observed reduction in triple negative breast cancer growth by Ang-(1-

7) corroborates the anti-proliferative effects of the heptapeptide in lung cancer

cells and lung tumors (60;146). Furthermore, the Ang-(1-7)-mediated reduction

in tumor growth may represent the molecular mechanism for the reported anti-

cancer effects of ACE inhibitors (50;52) which increase circulating levels of Ang-

(1-7) (14). The growth inhibitory actions of Ang-(1-7) are mediated by an AT(1-7)

receptor which is a 7 transmembrane spanning, G protein-coupled receptor

encoded by the mas gene (34;35). Mas is present on both MDA-MB-231 and

HCC38 human breast cancer cells, suggesting that it mediates the anti-

proliferative response to Ang-(1-7) in breast cancer cells. Our data suggests a

distinct and novel mechanism of growth inhibition in which Ang-(1-7) activates its

specific receptor to up-regulate DUSP1/MKP-1 to dephosphorylate and

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deactivate MAP kinases, leading to inhibition of cell growth and cell survival.

This suggests that Ang-(1-7) should be considered as a chemotherapeutic agent

in breast cancer, especially in the treatment of triple negative breast cancer

where there is a lack of targeted treatments for the patients that suffer from this

aggressive subtype of breast cancer.

Ang-(1-7) Inhibits Tumor Angiogenesis in Lung Tumor Xenografts and in Triple Negative Breast Orthotopic Tumors

We show for the first time that Ang-(1-7) reduces the growth of human

lung tumor xenografts and orthotopic triple negative breast tumors by a decrease

in blood vessel density. Ang-(1-7) reduced CD34 immunoreactivity in tumor

sections of Ang-(1-7)-treated animals, suggesting that one mechanism by which

the heptapeptide decreases tumor cell proliferation is through a reduction in

angiogenesis. In agreement, we demonstrated that Ang-(1-7) inhibits tubule

formation as well as reduces the number of blood vessels in the developing

CAM. More important, we found that Ang-(1-7) markedly reduced VEGF in both

lung tumor xenografts and breast orthotopic tumors as well as the parent lung

and breast cancer cells, suggesting that a decrease in the production of this

potent growth factor is involved in the anti-angiogenic response to the

heptapeptide.

A reduction in angiogenesis by Ang-(1-7) is supported by results from

several previous studies. Captopril, an anti-hypertensive drug that increases

circulating Ang-(1-7),(14) inhibited neovascularization in the rat cornea induced

179

by basic fibroblast growth factor (bFGF) and prevented capillary endothelial cell

migration by blocking chemotaxis towards an inducer of angiogenesis (130).

Ang-(1-7) also reduced angiogenesis in a murine sponge model of angiogenesis,

a technique representative of the formation of new blood vessels from pre-

existing ones during wound healing (25); the heptapeptide reduced hemoglobin

content, blood flow, and proliferative activity, as compared to vehicle. The anti-

angiogenic effect of Ang-(1-7) in this model was regulated by an Ang-(1-7)-

specific receptor,(25) in agreement with our identification of an Ang-(1-7)

receptor on endothelial cells from canine coronary artery and bovine aortic

endothelial cells (8;173;174). Further, the anti-angiogenic response was blocked

by pre-incubation with either aminoguanidine or NG-nitro-L-arginine methyl ester

(L-NAME), indicating that the response is mediated by nitric oxide (NO) (31).

Ang-(1-7) stimulates the release of NO from bovine aortic endothelial cells and

causes the vasodilation of blood vessels through an endothelial release of NO.

While the studies by Machado et al. (25;31) are in a model of angiogenesis

during wound healing, we now show that Ang-(1-7) inhibits angiogenesis that

occurs during tumor formation to supply necessary nutrients for tumor growth.

Mechanisms of Ang-(1-7) Inhibition of Tumor Angiogenesis Triple Negative Breast Orthotopic Tumors

The influence of angiogenesis on breast tumor growth follows the general

parameters that have now become dogma in the cancer field. Breast tumors

remain dormant and in situ without neovascularization; an alteration in the

balance of pro- and anti-angiogenic factors stimulates angiogenesis and

180

increases tumor growth (175). We investigated the anti-angiogenic effects of the

heptapeptide in breast cancer, based upon our initial studies showing that Ang-

(1-7) inhibits angiogenesis to reduce lung tumor growth. Subcutaneous

administration of Ang-(1-7) caused a significant reduction in growth and volume

of triple negative orthotopic tumors. Immunohistochemical analysis of tumor

sections using the CD34 endothelial cell marker demonstrated that treatment of

animals with Ang-(1-7) reduced vessel density when compared to sections of

tumors from animals treated with saline. This confirms our previous findings that

Ang-(1-7) inhibited lung tumor angiogenesis to reduce tumor growth. Our results

indicate that Ang-(1-7) treatment caused a significant reduction in VEGF,

suggesting that the observed reduction in tumor angiogenesis is due in part to

the inhibition of this pro-angiogenic cytokine.

VEGF is regulated at the level of transcription and translation by HIF-1α.

The activation of VEGF transcription occurs when HIF-1α binds to the hypoxia-

response element (HRE) upstream of the VEGF gene (176). HIF-1α also

stimulates VEGF synthesis by stimulating internal ribosome entry site (IRES)-

mediated translation; HIF-1α promotes the IRES-mediated translation of VEGF

during hypoxic-dependent and independent conditions (176;177).

Our studies showed that Ang-(1-7) reduced VEGF protein but not VEGF

mRNA in the triple-negative breast tumors. The molecular mechanism for this

regulation is not known but it is possible that HIF-1α is involved. Ang-(1-7) may

181

inhibit activation of HIF-1α through inhibition of activating kinases, such as MAP

kinases. It was demonstrated that ERK1 and ERK2 directly phosphorylate HIF-

1α to increase expression (154;178). Alternatively, Ang-(1-7) could stabilize

oxygen levels in the tumor microenvironment. NADPH oxidase generated

superoxide inhibits the activity of Von Hippel-Lindau (VHL) tumor suppressor

gene causing stabilization of HIF-1α. (179). Ang-(1-7) reduced reactive oxygen

species through an inhibition of NADPH oxidase (180). This suggests that Ang-

(1-7) may stabilize levels of oxygen tension in the tumor microenvironment

allowing activation of VHL to target HIF-1α for proteosomal degradation and

subsequently causing a reduction in VEGF. As discussed below, Ang-(1-7)

reduces PlGF, allowing circulating VEGF to bind Flt-1 which may serve as a

“trap” to decrease VEGF protein; this would also explain a reduction in protein

but not mRNA as observed in our lung cancer studies.

We previously demonstrated that Ang-(1-7) treatment decreased VEGF

mRNA in human lung tumors; however we did not observe a change in VEGF

gene expression in our studies in triple-negative breast cancer tumors. The half-

life of VEGF mRNA is significantly increased due to the presence of three

sequence elements within the 5’ and 3’ untranslated regions (181). Binding of

hypoxia-induced stability factor (HuR) in this region increases the half-life of the

VEGF transcript three-to eight fold (181;182). The mechanism for the induction

of these stabilizing factors is not known. However, these hypoxia-induced

stability factors may be expressed in the breast tumor tissue in our experiments,

182

overcoming the reduction of VEGF mRNA by Ang-(1-7) that we observed in our

lung cancer studies.

Treatment with Ang-(1-7) also caused a reduction in PlGF in our triple-

negative breast tumors. PlGF is a member of the VEGF family of cytokines

which has a 53% sequence homology to VEGF. Although the regulation of PlGF

was initially characterized in the placenta, it is also elevated during ischemic

conditions, inflammation, wound healing and hypoxic conditions (113). An

increase in circulating PlGF is correlated with tumor, stage, vascularity and

recurrence (113). PlGF expression is associated with an increase in pathological

angiogenesis, through activation of the VEGFR-1 or potentiation of the pro-

angiogenic effects of VEGF through VEGFR-2 (183). PlGF preferentially binds to

Flt-1 which has one-tenth of the tyrosine kinase activity of VEGFR-2 (184). It is

well-established that most of the angiogenic effects of VEGF are mediated

through VEGFR-2 (98). Therefore, if Ang-(1-7) treatment inhibits PlGF

expression, the high binding affinity of VEGF to Flt-1 may serve as a “trap” to

decrease VEGF to reduce its pro-angiogenic effects. This could also explain a

reason for reduction of VEGF protein expression and not mRNA with Ang-(1-7)

treatment in our experiments in triple-negative orthotopic tumors. However,

conflicting evidence demonstrates that Flt-1 can stimulate endothelial vessel

maintenance, growth and migration (183;184). Flt-1 is also essential in

development; Flt-1 (-/-) mice are embryonic lethal as a result of disorganization of

blood vessels and abnormal vasculature development (185). PlGF and VEGF

183

form heterodimers and work synergistically to induce angiogenesis, suggesting

that there is some overlap in the mechanisms which regulate both the activation

and inhibition of these pro-angiogenic factors (112). This suggests that the

observed reduction in PlGF may be a consequence of the Ang-(1-7)-mediated

decrease in VEGF. Alternatively, the reduction in PlGF by Ang-(1-7) may be

independent of its effect on VEGF. Even though PlGF is increased during

hypoxia, its expression is not mediated through activation of HIF-1α, indicating

that Ang-(1-7) would not inhibit PlGF expression through a HIF-1α-related

mechanism. Conversely, Ang-(1-7) could directly reduce the expression of PlGF

through the inhibition of MAP kinase activity. Analysis of the promoter region of

PlGF identified putative consensus sequences for known hypoxia-responsive

regulatory sites, such as Sp1-like sites (186), which are activated by ERK1 and

ERK2 (154;187). This indicates that a reduction in MAP kinase activity by Ang-

(1-7) could be responsible for the attenuation of PlGF expression.

A reduction in VEGF and PlGF following treatment with Ang-(1-7) could

also result from an increase in the soluble form of Flt-1 (sFlt-1). sFlt-1, produced

by alternative splicing (185;188), contains the six N-terminal immunoglobin-like

extracellular ligand-binding domains but does not include the transmembrane-

spanning region or the intracellular tyrosine kinase domains(188) resulting in a

receptor with high affinity for both VEGF and PlGF but no activation of

downstream signaling pathways (98). sFlt-1 is expressed in 85% of breast

carcinomas (184) and is significantly correlated with VEGF. Tumors in which the

184

sFlt-1 levels exceeded VEGF by 10-fold had a markedly favorable

prognosis,(184) suggesting that levels of both VEGF and sFlt-1 have a higher

prognostic value when considered together. A recombinant sFlt-1 effectively

bound VEGF and inhibited the VEGF-associated mitogenic activity (189) and

injection of a recombinant adeno-associated virus-2 (rAAV) encoding the human

soluble sFlt-1 increased survival and caused a significant reduction in ovarian

tumor xenografts growth (189). The estrogen-dependent breast cancer cell lines

MCF-7 expresses more sFlt-1 protein than the estrogen receptor negative MDA-

MB-231 (triple negative) cell line,(190) suggesting that maneuvers to increase

sFlt-1 levels might be beneficial in the treatment of triple negative breast cancer.

Future studies will determine whether the reduction of VEGF and PlGF by Ang-

(1-7) involves an increase in sFlt-1.

We showed that Ang-(1-7) reduces MAP kinases activities in both lung

cancer cells as well as in triple negative breast cancer cells and tumors. In triple

negative breast cancer cells and tumors, the reduction in ERK1/ERK2 activities

was associated with the Ang-(1-7)-mediated up-regulation of DUSP1 to

deactivate MAPK activities. Since ERK1 and 2 can directly or indirectly increase

VEGF and PlGF expression, we investigated whether the up-regulation of

DUSP1 is associated with the Ang-(1-7)-mediated reduction of VEGF and PlGF.

MDA-MB-231 cells were transfected with siRNA’s to DUSP1 and to cyclophillin,

as a control. Ang-(1-7) reduced VEGF and PlGF production in cells transfected

with the control cyclophillin siRNA. However, when cells were transfected with

185

an siRNA to DUSP1 and treated with Ang-(1-7), the heptapeptide did not reduce

either VEGF or PlGF, indicating that Ang-(1-7) binds to its unique receptor to up-

regulate DUSP1, inactivate ERK1/ERK2 and reduce the production of these pro-

angiogenic cytokines. MAP kinase activation not only promotes expression of

pro-angiogenic factors but also down-regulates endogenous inhibitors of

angiogenesis, such as thrombospondin-1 (TSP-1) (191). Treatment of

endothelial cells (HUVEC) with low concentrations of NO (0.1 µM) activated MAP

kinases, down-regulated TSP-1, and reduced proliferative response in the

endothelial cells. Increasing NO (100 µM) up-regulated DUSP-1 which was

associated with a reduction in MAP kinase activities and re-accumulation of TSP-

1. These results suggest a role for DUSP-1 in the the negative regulation of

angiogenesis by Ang-(1-7).

The effects of PlGF and VEGF on carcinogenesis are intricate and are

associated with both the tumor microenvironment as well as the expression of

their receptors in cancer cells. Our data indicates that the reduction of these

cytokines is associated with a reduction in tumor endothelial vessel density.

Experiments in the MDA-MB-231 parent cell line indicated that Ang-(1-7) caused

the receptor-mediated reduction of VEGF and PlGF. Moreover, Ang-(1-7)

receptor blockers prevented the inhibition of angiogenesis by the heptapeptide in

the chicken embryo. We identified the AT(1-7) receptor mas in the triple negative

cell line MDA-MB-231 as well as in endothelial cells. Taken together, these

results suggest that Ang-(1-7) inhibits angiogenesis to reduce tumor growth and -

186

should be considered as a targeted chemotherapeutic agent for the treatment of

triple-negative breast cancer.

Mechanisms of Ang-(1-7) Inhibition of Tumor Angiogenesis in Lung Cancer Xenografts

VEGF stimulates angiogenesis through a variety of mechanisms including

an increase in vascular permeability, induction of endothelial cell migration and

division, promotion of endothelial cell survival, and stimulation of cell proliferation

(192). A marked decrease in VEGF was observed in the tumors from mice

treated with Ang-(1-7) as compared to tumors from control animals, suggesting

that the heptapeptide attenuates tumor angiogenesis in the lung by reducing

VEGF. The precise molecular mechanism(s) for the Ang-(1-7)-mediated

decrease in VEGF is not known. In our lung tumor xenografts, VEGF mRNA was

also reduced, suggesting that Ang-(1-7) activates signaling pathways to inhibit

VEGF transcription. The VEGF promoter is regulated by a variety of factors;

however, the major regulator of VEGF gene transcription in lung cancer cells is

the hypoxia-induced factor HIF-1 (98). HIF-1 is increased in response to hypoxia

or conditions of low oxygen tension. Low oxygen tension in tumor cells located a

distance away from blood vessels results in the production and release of VEGF

to initiate angiogenesis. As a consequence of the anti-proliferative effects of

Ang-(1-7), metabolic activity and oxygen consumption may be reduced to modify

the tumor microenvironment and stabilize the HIF heterodimer.

187

In addition, HIF-1α expression is regulated by other pathways. MAP

kinases are essential for the direct phosphorylation and activation of HIF-1α

which in turns induces VEGF expression (154;178). Continuous activation of

ERK1 and ERK2 inhibits apoptosis, controls proliferation of endothelial cells, and

promotes VEGF expression by stimulating the activator protein-2 (AP-2/Sp-1)

complex on the VEGF promoter (154). We previously showed that Ang-(1-7)

inhibits the activities of ERK1 and ERK2 in lung cancer cells (60) and VSMCs

(30) suggesting that the observed reduction in VEGF mRNA and protein may be

due to inhibition of MAPK signaling. Other growth factors also increase HIF-1α,

the HIF-1 regulatory subunit. If HIF-1α is not regulated by Ang-(1-7) treatment,

VEGF may be controlled at transcription by other modulators, such as epidermal

growth factor, transforming growth factor β, or bFGF (193). In addition, VEGF

expression is regulated at transcription by RNA stability, at translation by mRNA

capping proteins, or at post-translation, by glycosylation (193). The precise

molecular mechanism(s) for the Ang-(1-7)-mediated decrease in VEGF is

currently under investigation.

In a previous study, we observed that administration of Ang-(1-7)

decreased lung tumor growth and volume by more than 30% while tumors of

mice infused with saline continued to grow 2.5-fold at the end of the 28 day

treatment (146). The decrease in tumor size caused by Ang-(1-7) was

associated with a reduction in COX-2, suggesting that the heptapeptide may

inhibit pro-inflammatory prostaglandins that promote lung tumor growth. Over-

188

expression of COX-2 is associated with the initiation of angiogenesis, through the

production of both prostaglandins as well as endothelial cell growth factors, and

COX-2 inhibitors block this process (194;195). COX-2 inhibitors suppress the

expression and secretion of the metalloproteinases MMP-2 and MMP-9,

suggesting that COX-2 also promotes tumor angiogenesis and tumor invasion.

In addition, the downstream products of COX-2 stimulate VEGF to promote

angiogenesis (194). Taken together, these studies suggest that the decrease in

angiogenesis by Ang-(1-7) may be due in part to a reduction in COX-2 mRNA

and protein.

The proliferation of endothelial cells is a crucial step in the angiogenenic

process. Endothelial cells are stimulated by tumor-released growth factors to

migrate and divide at the tumor site, ultimately forming blood vessel tubes

stabilized by smooth muscle cells. We demonstrated that Ang-(1-7) inhibited

endothelial cell tubule formation using a Matrigel assay. The inhibition of

angiogenesis by Ang-(1-7) may be due in part to its anti-proliferative properties to

reduce critical endothelial cell division and prevent the formation of vessels.

Ang-(1-7) also causes the receptor-mediated reduction in the proliferation of

VSMCs in vitro and in vivo (1;20;39) through stimulation of prostacyclin

production and activation of the cAMP-dependent protein kinase. The reduction

in vessel density observed in the human lung tumor xenografts or in the CAM

may be due to a loss in structural support provided by VSMCs. Alternatively, the

heptapeptide may block tube formation by inhibiting endothelial and/or vascular

189

smooth muscle cell migration. Studies are currently underway to delineate the

direct effect of Ang-(1-7) on the cellular components forming the vessel tube.

Cancer is the second leading cause of death in the United States with a

projected 1,437,180 new cancer cases and 565,650 deaths. Although progress

in research has resulted in the reduction of disease mortality rates, it is urgent to

find novel therapeutics that result in the reduction of disease morbidity and

increase in survival rates. Since the Ang-(1-7) mediates biological effects by

activation of a unique, G protein-coupled receptor, mas, this heptapeptide may

serve as an effective, targeted chemotherapeutic agent for cancer.

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Scholastic Vitae

David R. Soto Pantoja dsotopan@wfubmc.edu

Business: Hypertension and Vascular Research

Center Wake Forest University School of Medicine

Medical Center Boulevard Winston-Salem, NC 27157-1032 (336) 716-3810

Personal Information:

Birthplace: Arecibo, Puerto Rico, USA

Birthdate: May 3, 1980

Education: 2008 to present Doctor of Philosophy Molecular Genetics and Genomics Program Dr. Ann Tallant, Advisor Wake Forest University School of Medicine Winston-Salem, NC 2006 to 2008 Doctoral Candidate

Molecular Genetics & Genomics Program Wake Forest University School of Medicine Winston-Salem, NC

2004 to 2006 Biomedical Graduate Student

Molecular Genetics Program Wake Forest University School of Medicine Winston-Salem, NC

2002-2004 Post-baccalaureate Research Education

Program Wake Forest University School of Medicine Winston Salem, NC

1998-2002 University of Puerto Rico

Mayaguez, PR B.S. Biology, Cum Laude

Employment:

209

2001 Stephen Dellaporta Laboratory Undergraduate Research Student Yale University New Haven, Connecticut

210

1999-2000 Office Assistant MARC/NIH-UPR Mayaguez, Puerto Rico

Other Professional Appointments and Activities: 2007 Critical issues in tumor microcirculation,

angiogenesis and metastasis Harvard Medical School Boston, MA

2002 Dr. Garcia Rinaldi Foundation

Summer Internship Mayaguez, PR

2001 Analysis of Mitochondrial DNA of Puerto

Rican residents Field Research Assistant University of Puerto Rico

2000 Pathology Department Summer

Program in Molecular Medicine for Undergraduate Students Yale University

1999 to 2002 Food Microbiology Laboratory

Undergraduate Research Assistant University of Puerto Rico

Professional Memberships: 2004 - present Member, Wake Forest University

Graduate Student Association 2006 - present Associate Member

American Association for Cancer Research 1999 - 2002 Member, BBB National

Biological Honor Society 2000 - 2002 Howard Hughes Research

Experiences and Training Opportunities (RETO)

Honors and Awards: 2008 Silver Award 16th Annual Residents and

Fellows Research Day

211

Wake Forest University School of Medicine, Winston-Salem, NC

2008 Lucy Robins Fellowship Award

Wake Forest University School of Arts & Sciences, Winston Salem, NC

2008 Best Poster Women’s Center of Excellence Research Day Wake Forest University School of Medicine Winston-Salem, NC

2006 AACR Minority Scholar Award in Cancer Research

2005 FASEB MARC Program Travel Award Association for Biomolecular Facilities Annual Meeting Savannah, GA

2004 Gold Award 12th Annual Resident’s and Fellow’s Research Day Wake Forest University School of Medicine Winston-Salem, NC

2002 Frank G. Brooks Award for

Excellence in Student Research Caribbean Convention National BBB Honor Society.

1999 BBB Leadership Award

BBB Biological Honor Society Mayaguez, PR

2000 Leo of the Year

Leo Club, Arecibo, PR

1998 4th Place Environmental Sciences, 49th Intel

International Sciences and Engineering Fair Forth Worth, TX

Research Funding: 2006 - present Department of Defense breast cancer pre-

doctoral traineeship award

212

Directory Listings: 1998 United States Achievement Academy

National Awards, Vol. 148 Community Activities: 2003 - present Stroke Education towards Minorities,

Volunteer Wake Forest School of Medicine

2004 Forsyth County Science Fair Judge 2004-06 Mentored High School Student

Math and Science Education Network (MSEN) Winston-Salem, NC

2002-to present Share the Health Committee,

Wake Forest School of Medicine Winston-Salem, NC

Poster Presentations: 2008 Era of Hope Meeting Ang-(1-7) inhibits the growth of human triple

negative breast tumors in an orthotopic mouse model

Baltimore, MD 2008 AACR Annual Meeting Ang-(1-7) inhibits growth of human

triple-negative breast tumors in an orthotopic mouse model San Diego, CA

Forty years of metastasis research: Angiotensin-(1-7) reduces lung cancer cell A symposium in honor of Dr. Isaiah Fidler growth through the inhibition of inhibition of angiogenesis MD Anderson Cancer Center Houston, TX 2005 AACR Annual Meeting Inhibition of angiogenesis by angiotensin-(1-7)

Washington, DC 13th Fellows and Residents Research Day Is Ang-(1-7) an inhibitor of angiogenesis? Wake Forest University School of Medicine Winston-Salem, NC 12th Fellows and Resident’s Research Day Inhibition of human breast cancer

213

cell growth by angiotensin-(1-7) Wake Forest University School of Medicine Winston-Salem, NC

2004 AACR Annual Meeting Inhibition of human breast cancer

cell growth by angiotensin-(1-7) Orlando, FL 11th Fellows and Residents Research Day Effect of angiotensin-(1-7) in human

breast cancer cell growth Wake Forest University School of Medicine Winston-Salem, NC

PREP Research Day Effect of angiotensin-(1-7) in breast cancer

cells Wake Forest University School of Medicine Winston-Salem, NC

2002 National Convention Toxigenic potential of S.aureus BBB Honor Society in Puerto Rican artisanal

dairy products San Antonio, TX Bio's 2002 "Escherichia coli in vitro

transcription/translation system" University of Puerto Rico Mayaguez, PR 2002 ACS, Junior Technical Meeting "Escherichia coli in vitro

transcription/translation system" San Juan, PR Poster Discussion: San Antonio Breast Cancer Symosium Angiotensin-(1-7) inhibits triple negative tumor growth through the inhibition of angiogenesis and a reduction in Placental Growth Factor (PlGF) Oral Presentations: 2008 Era of Hope Meeting Ang-(1-7) inhibits triple negative

breast cancer in an orthotopic mouse model Baltimore, MD Abstracts:

214

Soto-Pantoja DR, Tallant EA, Gallagher PE. Inhibition of human breast cancer cell

growth by angiotensin-(1-7). Proceedings of the 2004 meeting of the American

Association for Cancer Research, March, 2004.

Tallant EA, Soto-Pantoja DR, Gallagher PE. Inhibition of human breast cancer cell

growth by angiotensin-(1-7). 2004 Susan G. Komen Breast Cancer Foundation

Mission Conference “Pathways to a Promise”, June, 2004.

Soto-Pantoja, DR, Gallagher PE and Tallant EA. Inhibition of angiogenesis by

angiotensin-(1-7). Proc Amer Assoc Cancer Res, Volume 47, 2006.

Gallagher PE, Soto-Pantoja DR and Tallant EA. Angiotensin-(1-7) inhibits

angiogenesis by reducing endothelial cell growth. Hypertension 2007

(Proceedings of the Council for High Blood Pressure Research), published online.

Soto-Pantoja DR, Gallagher PE and Tallant EA. Angiotensin-(1-7) inhibits the growth of

human triple negative breast tumors in an orthotopic model. Proceedings of the

99th Annual Meeting of the American Association for Cancer Research; 2008 Apr

12-16; San Diego, CA. Philadelphia (PA): AACR; 2008. Abstract 2416.

Menon J, Miller MS, Soto-Pantoja DR, Callahan MF, Tallant EA, and Gallagher PE.

Angiotensin-(1-7) reduces lung tumor incidence in a novel bitransgenic Ki-rasG12C

mouse model of lung tumorigenesis through a reduction in cyclooxygenase-2.

Proceedings of the 99th Annual Meeting of the American Association for Cancer

Research; 2008 Apr 12-16; San Diego, CA. Philadelphia (PA): AACR; 2008.

Abstract 4654.

215

Soto-Pantoja DR, Menon J, Gallagher PE, and Tallant EA. Angiotensin-(1-7) inhibits

the growth of human triple negative breast tumors in an orthotopic model.

Proceedings of the 2008 Era of Hope Meeting, Baltimore, MD.

Gallagher PE, Soto-Pantoja DR and Tallant EA. Angiotensin-(1-7) reduces endothelial

cell growth and migration and increases VEGI to inhibit angiogenesis.

Hypertension 2008 (Proceedings of the Council for High Blood Pressure

Research), in press.

Publications: Tallant EA, Menon J, Soto-Pantoja DR, and Gallagher PE. Angiotensin peptides and

cancer. In: Handbook of Biologically Active Peptides (Kastin AJ, editor) Elsevier,

San Diego, pp. 459-465, 2006.

Menon J, Soto-Pantoja DR, Callahan MF, Cline JM, Ferrario CM, Tallant EA and

Gallagher PE. Angiotensin-(1-7) inhibits growth of human lung adenocarcinoma

xenografts in nude mice through a reduction in cyclooxygenase-2. Cancer

Research 67, 2809-2815, 2007

Soto-Pantoja DR, Menon J, Gallagher PE and Tallant EA. Angiotensin-(1-7) inhibits

tumor angiogenesis in human lung cancer xenografts with a reduction in vascular

endothelial growth factor. In revision in Molecular Cancer Therapeutics.

Soto-Pantoja DR, Menon J, Gallagher PE and Tallant EA. Angiotensin-(1-7) inhibits

growth of triple negative breast cancer in a mouse orthotopic model. To be

submitted to Cancer Research November 2008

216

Menon J, Miller MS, Soto-Pantoja DR, Callahan MF, Tallant EA, and Gallagher PE.

Angiotensin-(1-7) reduces lung tumor incidence in a novel bitransgenic Ki-rasG12C

mouse model of lung tumorigenesis through a reduction in cyclooxygenase-2. (In

preparation).

Soto-Pantoja DR, Gallagher PE and Tallant EA. Angiotensin-(1-7) inhibits the growth of

triple negative breast cancer through the reduction in angiogenesis. (In preparation).

217

VITA

David Ricardo Soto Pantoja was born on May 3rd, 1980 in Arecibo, Puerto Rico.

He did his undergraduate work at the University of Puerto Rico, Mayaguez

Campus, and received a Bachelor of Science, Cum Laude in Biology. After

college he moved to Winston-Salem were he was a member the Wake Forest

University School of Medicine Post-baccalaureate Research Education Program

(PREP) were he joined the laboratory of Dr. Ann Tallant. After completion of this

training program he formally joined the student body of the Wake Forest

University Graduate School of Arts and Sciences as a member of the Molecular

Genetics & Genomics Program and initiated his dissertation under the guidance

of Drs. Ann Tallant & Patricia Gallagher, where he pursued his interest in

molecular biology and discovery of novel anti-cancer therapeutics.