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Li Long. ReguhImcm nf Twnor CeH Invmion unci Meranatir by rhe Tvpe / Insuiin-like Growrh Feror Receptor (IGF- f R)

rn Regulation of Tumor Cell Invasion and Metastasis

by the Type 1 Insulin-like Growth Factor

Receptor (IGF- IR)

Li Long

Department of Surgery, Division of Surgical Research

McGill University

Montreai Canada

Jdy, 1997

A Thesis submitted to the Faculty of Graduate Studies and Research

in partial fulfillment of the requirements of the degree of Doctor of

Philosophy

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Candidates have the option of including, as part of the tksis, the text of one or more

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or more published papers. these texts must be b o d as an integral part of the thesis.

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be integrated

The thesis m u t still confonn to al1 other requirements of the "Guidelines for Thesis

Preparation". The thesis n w t include= A Table of Contents, an abstract in English and

French, an introduction which clearly States the rationale and objectives of the study, a

cornprehensive review of the literature, a final conclusion and S M M I I ~ ~ , and u thorough

bibliography or reference lis?.

Additioml merial maut be provided where appropriate (e.g. in appendices) and in

suflcient detail ro allow a clear anà precise judgment to be made of the importance md

originality of the research reported ùt the thesis.

In the case of manuscripts co-authored by the candidate and others, the candidote is

required to d e an exphkit sfatement Ur the daesis as &O who contdiuted to such work

and to what extent Supervisors must anest to the accuracy of such statements ar the

doctoral oral defense. Since the task of the examiners is made more dificuit in these

cases, it is in the candidate's interest tu make peflectly clear the responsibilities of al1 the

authors of the co-authured papers. Under no circumstances can a co-author of any

component of such a thesis as an examiner for that thesis.

In accordance with the above guidelines, 1 have decided to include the following published

papers as part of the body of the chesis:

Long, L., Nip, J., and Brodt, P. Paracrine growth stimulation by hepatocyte-

denved IGF-1: A regulatory mechanism for carcinoma cells metastatic to the

Iiver. Cancer Research. 54: 3732-3737, 1 994.

Long, L., Rubin, R., Baserga, R., and Brodt, P. Loss of The metastatic

phenotype in murine carcinoma cells expressing an antisense RNA to the

insulin-like growth factor receptor. Cancer Research. 55: 1006-1 009, 1995.

These two papers are reproduced h m Cancer Research by copyright permission of The

Amencan Association for Cancer Research. I am responsible for al1 of the experimental

work and anaiysis carrieci out in these two aforementioned papers with the exception of

a construction of plasmid vectors expressing IGF-IR cDNA in the sense or antisense

orientations. AU of the work was performed in the Iaboratory of Dr. Pnina Brodt

(Deparunent of surgery, Division of Surgical Research, McGïii University).

With the dream that one day I would be able to contribute to the fight against one of the

deadliest enemies of human health - cancer, 1 applied for Ph.D. snidies in the Department

of Surgery, Division of Surgical Research, McGill University. First and foremost, 1 would

iike to gratefuliy acknowledge the invaluable supervision, personai guidance, endless

encouragement and financial support given by Dr. Pnha Brodt. As a mentor, she has

generously given me the opportunity to develop my research skills, detailed suggestions

and guidance with great patience in ai l aspects of m y study. She has dways been available

and wiliing to assist me when 1 encountered any type of problem, research or otherwise,

despite her busy schedule. Dr. Brodt's role in m y professional development bas been

paramount and is highly appreciated.

A special thanks goes to my colleague, Dr. John Nip, who has generously aven me

enormous help and assistance in the fields of molecular biology, biochemistxy and

computer science when we studied togetber.

1 would Like to express my sincere appreciation to Drs. Raphael Rubin and Renato

Baserga for providing us the precious plasmid vectors required for portions of this study. -

for offenng their insight through discussions and for spending time to read and comment

on our rnanuscnpts.

1 would iïke to thank Mrs. Lucia Faüavoiiïta for her help in teaching me the numerous

tec hnical me thodologies and giving me sound practicd advice and generous assistance in

rnatters of research.

1 would Lice to acknowledge my colleagws and f'riends in the laboratory Dr. Hua Ling, for

her assistance in the field of molecular biology and her encouragementv and Dr. Margaret

Durko. Dr. Jian Wang, M s Roya Navab, Dr. Keguan Chen, Dr. Takayoki Assao and Mrs.

Grazieiia Vaknte for Liberal assistaoce and thoughtfùi discussions. As weU, the genemus

assistance, endless encouragement and invaluable fkiendship given by aU other members of

this Department, especiaüy Dr. Julius Gordon, Mrs. Nina Hassan , Mrs. Irene

Sidorenko, Mrs. Am Gordon and my fiends in Donner Building Mrs. Pat Guida . Ms. Linda Bazinet and Mr. Alberto Rodrigues, are appreciated. 1 have overcome mauy

difficulties through their help. Parties, dinners and cards are beautifid mernories that will

reside in my mind forever.

1 am grateful to Dr. B. R. Zetter (Department of surgery, Harvard Medical School,

Boston, MA), Dr. C. B. Srikant (Department of Medicine, McGii University, Montreal).

Dr. B. Massie (The Biotechnology Research Institute, National Research Council of

Canada, Montreai) and Dr. G. Karpati (Neuromuscular Research Group. Montreal

Neurological Institute, McGiU University, Montreai) for giving me the oppominity to

work in their labratories during some aspects of my study.

a 1 would like to thank Dr. Abdel M. Khatib for his kind assistance in the translation of my

abstract.

FmaUy, a speciai thanks to my wife Sining Wang, to my mother Mrs. Chunying Jiang, to

my parents in-law Mr. Kuiye Wang and Mrs. Huaming Liu, and to my son Randy Long for

their wholehearted support and encouragement.

Original Contributions to the Body of Knowledge

The major novel findings of the p m n t study are as foiiows:

1 . Liver derived IGF-1 is a major mitogmic and chernotactic factor for liver

metastatic cells;

2. Antisense mRNA to IGF-IR inhibits tumor ce11 gmwth and invasion

viîro and completely blocks metastasis in vn>o ; - 3. Overexpression of IGF-IR in a moderately metastatic tumot ce11 iine

increases the mitogenïc response to IGF-1 and celi invasion in vitro and

enables the cells to colonize the liver in vivo;

4. IGF-I is a major regulator of the expression of the Mr 72,000 type IV

collagenase (MMP-2, gelatinase A)

Abbreviations

DMSO

EGF

ECM

FN

HCM

IGF-1

IGF-IR

IGFBPs

1 s .

1.v.

MMP-2

MAb

PDGF

TIME's

TGF

Dimethyl sulfoxide

Epidermal growth factor

Extracellular matrix

Fibronectin

Hepatocyte conditioned medium

The type 1 insulin-like growth factor

Receptor for type 1 insulin-like jpwth factor

Insulin-like growth factor bindbg proteins

Intrasplenic

Intravenous

Matrix metalloproteinase 2

Monoclonal antibody

Platelet derived growth factor

Tissue inhibitor of metalloproteinases

Transforming growth factor

Table of Contents

.....................*......................... ....................*................*.. ..**.... A bstract .,., ..,... XVD

Chapter 1 . Molecular mediators of cancer cell invasion and metastasis . An

.......................................................... 1 . 1 . Introduction ............................................. -2

1.2. Growth factors and growth fa~tor receptors .............................................................. 3

......................................................... 1 .3 . The extracellular ma&-degradïng protehases 9

................................................................................ Cathepsins .................... .. 1 4

............................................................................. The metrix metaiioproteinases 15

....................................................................................................... Glycosidases 18

...................................................... ........... 1.4. The role of ceii adhesion molecules ... 1 9

........................................................................................ ............ In tegrins ... 20

.................................................................................................... The cadherins -22

............................ ...........*.... The immunoglobulin (Igs) supergene family ...... 24

..................................................................................... ................... Selectins .. 26

..................................................... .......................... 1.5. Angiogenesis and metastasis .... 28

..................................................................................................... 1.6. Motility factors -30

.............................................................................................................. 1.7. Summary 31

Chapter II . The insulin-like growtb factor 1 meptor: Structure, function and d e in

................................................................................................................ maügnancy 33

2.1. The type 1 insulin-like growth factor receptor (IGF-IR) ......................................... 34

................................................................................................. 2.2. The IGF-IR ligand -35

2.3. The IGF binding proteins ...... .. ............................................................................... 37

.................. ........................................................................... 2.4. IGF-IR signaling ,.. 39

2.5. Role of IGF-1 in cellular proliferation ...................................................................... 43

2.6. Role of IGF-I and IGF-IR in ceii death ................... ... ....................................... 45

........................................................................................ 2.7. IGF-IR and malignancy -45

............ Chapter III . Mahix met di op rote^ in cancer invasion and metastasis 48

.................................... The role of metalloproteinases in -or invasion and metastasis 52

Chapter IV . The d e of insuiin-lilre growth factor 1 in metastasis: Studies with the

................................................................................... Lewis lung carcinoma mode1 -56

4.1. Summary ....................................................................................... ....... 57 ...............

4.2. Sublines H-59 and M-27 of the Lewis lung carcinoma: Review of published data .... 58

Chapter V . The d e of insuün-iike growth factor 1 m p t o r system in cancer

me- (II): Enfimced invasion and liver-colonization in lung carcinoma c&

........................ .............. overexpressing the insulin-like growth factor 1 receptor .. 61

.............................................................................................................. 5.1. Overview -62

................................................................................................................. Introduction -64

.......................................................... ..................................... Materials and Methods .. -66

................................................................... .............................. Results .......... 7 1

.................................................................................................................... Discussion 73

Chapter VI . The role of iasulln-like growth factor 1 ~ e ~ e p t o r system in cancer

metastasis 0: Reguiation of Mr 72, 000 type N coïiageoase synthesis by the type 1

........................................................................ insulin-like growth factor receptor 82

................................................................................ ............................... Overview ...... 83

........................................................................................ ..................... Abstract ... 85

............................................ Materials and Methods .............................. .........., 88

.................................................................................................................... Results -92

................................................................................................................ Discussion 94

Chapter VIT . Tumors 8-59 and M-27 cek M e r in their rcsponses to IGF-I and

Merentially express other growth factors and receptors . Summ~ry of unpubbhed

................................................................................................................. results -102

....................................................................................................... 7.1. Introduction 103

....................................................................................... 7.2. Materials and Methods 103

............................................................................................................... 7.3. Results 107

........................................................................................................ 7.4. Discussion. 110

....................................................................................... C hapter VIII. Discussion 117

......... 8.1. Differential expression of growth factors and receptoa: tumor heterogeneity 118

.............. 8.2. The role of the IGF-IR/IGF-1 complex in cancer invasion and metastasis 120

..................................... ................................ 8.3. Suggestions for hiture research .... 127

.............................................................................................................. References 128

Li t5~n.v. Reg.uIuim r>/Tmar Ceff Invurü~n und Meranaris by ihc Tvpc 1 Inrufin-lik Gmwrh Fucmr Receprrw (IGF-1 RI

List of figures

Chapter IV

A ttached paper entitled " Paracrine growth stimulation by hepatocyte-derived

insulin-like gro wth factor4 A regulatory mechanism for carcinome cells

metastatic to the liver."

Finure 1. Bar graph showing the effects of conditioned media and growth factors on DNA

synthesis by the tumor ceh.

Figure 2. Line graph showing the dosedependent stimulation of H-59 ceiis by IGF-1.

Figure 3. Composite Line graph of inhibition and depletion of the mitogenic effect of

hepatocyte conditioned medium by a monocionai antibody to IGF-1.

Fiaure 4. Photograph showing the detection of IGF-1 in hepatocyte and lung tissue

conditioned media by Western blot analysis.

Figure 5. Scattered graph and autoradiograph composite demonstrate Scatchard analysis

of IGF-1 binding and Northem blot analysis for IGF-IR mRNA transcripts expresseci by

tumor cells.

Fipure 6. Autoradiograph and photograph composite showing the detections of KGF

binding proteins by Western ligand blot assay and Western blot assay.

Attached paper entitled "Loss of the metastatic phenolype in murine carcinoma

cells expressing an antisense RNA to the insulin-like growth factor receptor-'*

xiii

Figure 1. Autoradiograph and bar graph composite showing decreased IGF-IR mRNA

expression in tumor celis transfected by plasrnid vector canyîng an antisense sequence to

IGF-IR.

m u r e 2. Line and bar graph composite demonstrates the loss of response to IGF-1 and

hepatocyte couditioned medium in IGF-R antisense transfected ceiis.

m u r e 3. Line graph showing the growth of H-59 and IGF-IR antisense transfected cek

in vivo.

Chapter V

m u r e 1. M-27 c d s overexpressing IGF-IR proMerate in response to IGF-1 and HCM.

FiPure 2. Analysis of human IGF-IR expression in M-27 tnmsfectants by RT-PCR and

immunoprecipitation.

Figure 3. Increased invasiveness of M-27 cells overexpressing IGF-IR.

Chapter VI

Figure 2. Decreased invasiveness of H-59 ceils expressing IGF-IR antisense mRNA.

m u r e 2. The effect of modulation of IGF-IR expression on MW-2 mRNA production in

H-59 and M-27 celis - Analysis by RT-PCR

Figure 3. Western blot analysis of MMP-2 and TIMP-2 production in IGF-IR transfected

M-27 cells.

m u r e 4. Zymographic analysis of MMP-2 activity in M-27 ceils overexpressing IGF-IR.

xiv

Chapter VII.

Fiaure 7- 1 . Northern blot analysis of growth factors and receptors expressed in H-59 and

M-27 ceiis.

Figure 7-2. The chernotactic effect of IGF-1 on H-59 ceiis.

Figure 7-3. IGF-1 induced tyrosine phosphoryIation assessed by Western blot anaiysis.

Fiaure 7-4. IGF-I protects H-59 cells from apoptosis.

List of Tables

Chapter IV

Table 4-1. Summary of the phenotypic differences between H-59 and M-27 cells.

Chapter V.

Tabb 1. Increased anchorage-independent p w t & in tumor ceUs overexpressing IGF-IR.

Table 2. Enhanced liver-colonizing potentiai of M-27 ceik overexpressing IGF-IR.

xvi

Abstract

invasion and metastasis largely determine the clinical course of cancer. A better

understanding of the mechanisms required for metastases formation in secondary sites

will lead to the development of new and more successful tmument strategies. The present

work describes our resdts with a murine Lewis lmg carcinoma mode1 which consists of

two ce11 lines, Hg59 and M-27, with different patterns of metastasis in vivo. Using tbis

model, a positive coneiation was found b e e n the expression of Msulin-like growth

factor 1 receptor (IGF-Et) and the ability of the tumor celis to metastasize to the liver. The

highly invasive, highly metastatic, liver colonizing cell line H-59 expressed significantly

higher levels (5-fold) of IGF-IR than the poorly invasive M-27 cells which metastasize to

the lung only. When expression of IGF-IR in 8 5 9 cells was suppressed by transfection

with a plasmid vector expressing MiF-IR cDNA in an antisense orientation, the cells lost

m . their proliferative response to IGF-1 NI vitro , their ability to migrate in response to IGF-1

and their metastatic potential. To further study the role of IGF-IR in the process of liver

metastasis, IGF-IR was overexpressed in the Iung metastasizing M-27 cells by

transfection with a plasrnid vector expressing fil1 length human IGF-IR cDNA. The

stable transfectants had an cnhsnctd proliferative respome to IGF-1 and hepatocyte

conditioned medium ( K M ) and acquired an invasive potential as demonstrated in the

Matrigel invasion assay. When inoculated via the splenic/portal route in vivo, these celIs

but not the wild-type or mock-trmsfected cells gave rise to multiple liver nodules . TO M e r investigate the link between IGF-IR and invasion, metalloproteinase 2 (MMP-2)

expression in the tumor cells was investigated. It was found that the antisense h~nsfected

H-59 cells expressed significantly lower levels of MMP-2 as assessed by RT-PCR,

Western blot d y s i s and gelatin zymography. M-27 cells overexpmsing IGF-IR had a

marked increase in MMP-2 mRNA expression with a comsponding increase in the levels

and activity of the protein. Stimulation of these cells with IGF-1 resulted in increased

production of MMP-2 protein and increased gelatinolytic activity. In conclusion, our

results show that the IGF-IR/IGFII complex can modulate several cellular functions

which impact on the metastatic potential including growth, migration, invasion and fiver-

colonization. For tumors which an dependent on this system for proliferation, invasion

and metastasis, the IGF-IR could pmvide a specific target for effective anti-metastatic

tlierapy .

L'invasion et les métastases determinent cliniquement le développement et la

progression du cancer. La comprChension des mecanismes impliqués dans la

formation des métastases serait très utile a la mise en place de nouvelles

stratégies th6rapeutiques.

Ce travail décrit les résuitats obtenu in vivo avec le carcinome de poumons

de Lewis chez la souris, ceci en éhldiant les deux lignées cellulains modèles:

H-59 et M-27. Nos résultats montrent l'existence d' une corrélation positive

entre l'expression des récepteurs de Iïnsulin-like growth factor4 (IGF-IR)

et la capacité des œllules B envahir le foie par métastase.

Les œllules H-59, cellules invasives métastatiques, et envahissantes du foie,

expriment 5 fois plus & récepteurs IGF-IR que les cellules M27. qui

possèdent un faible pouvoir d'invasion et qui ne métastase que les poumons.

La transfection des cellules H59 avec un plasmide contenant de 1'ADNc des

rkcepteurs IGF-IR oriente dans un sens contraire (anti-sens), entraînanant la

suppression de l'expression des rCcepteurs IGF-IR, induit in vivo chez ces

cellules une perte de leurs capacitt de pmLif6rer et de migrer en présence de

lfIGF-1; et de leur pouvoir m6tastatique. Afin d'étudier le r61e du récepteur

de I'IGF-1 dans les processus des métastases du foie, ce dernier a et6 sur

exprimé dans les œllules M-27 B l'aide d'une transfection par un plasmi&

contenant I'ADNc complète du récepteur IGF-IR humain. Les cellules ;

trasfectées montrent une augmentation de leur capacitt de proliferer en

présence de lq1GF-I et du milieu conditionné des hépatocytes (HCM) et

acquièrent un pouvoir d'invasion. comme le &montre le test d'invasion du

Matrigel. L'inoculation in vivo de ces cellules au niveau de la veine porte

induit une augmentation des nodules du foie. M m de trouver la relation qui

existe entre les récepteurs IGF-IR et l'invasion , nous avons étudie

l'expression de la matria métallopmtéase 2 (MMP-2) dans les cellules

tumorales. Cette étude montre que les cellules H-59 transfectées n'expriment

que trés faiblement la MMP-2 comme le démontrent les technique de RT-

PCR, Western blot et la zymograpbie. Les œllules M27 qui sur expriment

les récepteurs IGF-IR présentent un taux très éleve des ARNm de la MMP-2

ainsi q'une activite augment6 de la protéine correspondante. La stimulation

de ces cellules avec de PIGF-1 se traduit par une augmentation de la

production de la MMP-2 et de son activité. En conclusion; nos résultats

montrent que le complexe IGF-IIIGF-IR peut moduler certaines fonctions

cellulaires qui ont un effet sur le pouvoir métastatique des cellules y compris

la croissance, la migration l'invasion et la colonisation du foie. Les Tumeurs

dont les cellules qui dependent de œ type de système pour leur prolif&ation,

invasion et métastase; le récepteur IGF-IR pourrait être une cible spécifique

m e pour la thérapie des métastases.

Molecular Mediators of Cancer Cell Invasion And Metastasis -

An Ovewiew

Li Long. Regdafion of Tumor Cell fnvusion a d l W e r ~ m i s by the Type I huuILn-lik Growih Factor Recepror (IGF-IU)

The clhical course of malignant disease is determined largely by the process of invasion

and metastasis. A better understanding of these processes is thetefore likely to lead to the

development of new and more successftl treatment strategies.

Over a century ago, Paget proposed that metastasis was due to the specific affhïty of

certain tumor cells (the "seed") for the milieu provided by certain organs (the "soil") (1)-

On the other han& the amtomid-mechanid theory of metastasis stresses the

importance of the vascular connections between the primary tumor and the secondary

site(s) of growth (2). Currentiy it is accepteci that both anatomid-mechanical and Seed-

soif factors play a role in the formation of metastases.

Metastasis is a cascade of Linked sequentiai steps involvïng multiple host-tumor

interactions (3-6). To successfuliy give nse to a metastatic colony, a ceil or group of

-or celis must detach h m the primay tumor, invade the local host tissue, enter the

cuculation, arrest at the distant vascular beâ, extravasate into the target organ interstitiun

and parenchyma, and proLiferate in the xcondary site. These processes are mediated by a

series of molecular interactions resulting h m disrupted positive and negative regdatory

mechanisms (7).

Invasion h m most primary epithelial tumors (carcinomas) requires disruption of the

basement membrane (8). This brings the tumor cells into contact with the underlying

Li Long. R4guIarion of T w o r CeII Inmaion and4CIe~ro1ü by rk Tipr f I~syIin-like Growtii Foccor Roccptor ffGF-IR)

a stroma and with parenchymal elements. The type of tissues encountered by the tumor

cells couId Vary dependîng on the tissue of origin of the malignant celis and theù pattern

of metastases, and rnay include connective tissue, muscle, bone, neuronal tissue and

epithelia (9). These tissues are impermeable to cells without prior alteration of their

composition and organhtion. Intravasation occurs at the level of lymph or blood

capillaries (9). Blood capillaries consist of an endothelium surromdeci by a continuous

basement membrane and pericytes; lymphatic capillaries M e r from blood capillaries

because they lack a basement membrane. When intravasaîion takes place at the level of

larger vessels, ~tumor ceils traverse additional layers of smooth muscle and connective

tissue. Tumor cells corne into contact with a multitude of serum factors and with cellular

elements cimilating in the blood or lymph. During extravasation, tumor celis transverse

the same structures encountered during intravasation. At each of the above-mentioned

steps, invasion may be accompanied by growth of the tumor cells, but growth and

invasion may also occur iadependentiy (10).

The following is a review of the factors and mechanisms involved in positive or negative

regdation of the process of metastasis.

1.2 Growth fwtom and gœwth factor receptora

The interaction of growth &tors, cytokines and hormones with their specific receptoa

triggers a cascade of intracellular biochemical signals, d t i n g in the activation and/or

0 repression of various subsets of genes (1 1). Genetic aberrations in growth factor signaling

Li Long. Regiriarion of Tumor Cd1 Invasion ami M e m w i t by the ïype I imfin-fi& Growth Factor Reeptor (IGF-IR)

a pathways are inexrricably Linked to developrnental abnormalities and to a variety of

chronic diseases, including cancer. Malignant cells mise as a resdt of a stepwise

progression of genetic events that include the dereguiated expression of growth factors,

theu receptors or components of their signai transduction pathways (1 1).

Growth factors enable ceils in the resting or Go phase to enter into and proceed through

the ceil cycle. The quiescent ceii must fkst advance into the G, phase of the ce11 cycle in

response to "~~mpetence~' ffactrs such as PDGF, procced through the GI phase, and then

become cornmitteci to DNA synthesis under the influence of "progression" f e r s such as

IGF-I and EGF (12-14). Growth factors and growth suppressers form a network of

regdatory signals in which either the overexpression of a positive signal or the decreased

expression of negative ones r e d t in a disturbance of ceil growth. These effects on

cellular proMeration are mediated via altered gene expression and protein synthesis (1 5).

Growth factors and their receptors have been implicated in tumor development (16-1 8)

including malignant transformation (19) and tumor progression (20). Expression of

PDGF and its receptor(s) has been documented in a high proportion of sarcomas as well

as giially denved neoplaîrns (21). in tissue culture, such tumor cells exhibit chronic

PDGF receptor activation, demonstrating a fimctional a u t o c ~ e loop induced by ligand

stimulation of receptoa produced by the same celi (22). Similarly, transforming growth

factor a (TGFa) is fkquentiy detected in carcinomas expressing high levels of the EGF

receptor (23). FGFs, such as basic fibroblast growth factor (bFGF) are upregulated in

a human melanoma cells which require bFGF to prolifetate (24). Among growth factor

receptors, the most fkequently implicated in human cancer have been memben of the

EGF receptor family. The EGF receptor gene is often overexpressed in squamous celi

carcinomas and glioblastomas (25). Similady, erbB-2, the oncogene which encodes a

receptor-like protein sharing high homology with EGF-R, is often overexpressed in

adenocarcinornas of the breast, stomach and ovary (26). Overexpression of either gene

under appropriate experimental conditions wnfm the t rdormed phenotype (27). The

erbB-3 gene is overexptessed in fertain breast carcinomas (28). Gene amplification or

overexpression of the mer gene encodiog the HGF receptor or of bek encoding a member

of the FGF family has been observed in human gastric carcinoma cell h e s (29).

Similarly, the ret gene encoding an EGF nceptor tyrosine kinase homolog is activated by

gene rearrangements in a large k t i o n of human thyroid carcinomas (30).

Many of these molecules have emerged h m oncogew cesearch as they were initially

found as products of oncogene-transformed cells. For example, the B-chain of platelet-

derived growth factor (PDGF) is encoded by the proto-oncogene C-sis (31). Several

oncogene pduc t s (e.g., hst or K-fg int-2, fjgf- and -6) share 4 0 4 % sequence

homology with basic fibroblast growth fector (bFGF) (32). The receptor for colony-

stllnulating factor 1 (CSF-1) is encoded by c-jmr (33). The v-erb-B-1 oncogene encodes a

tnincated receptor for e p i d e d growth fmor (EGF-R or p-c-erb-1). in which the

extracellular Ligand-binding domain is lacking, but the trammembme domain and an

intracelldar tyrosine-kinase domain is prrserved (34). Other oncogene products show at

Li Long. ReguIation of Tuaor Ce11 Illwuion and Merastasit by the Tjpe I InsnIin-lik G r o w l Factor Reccpror (IGF- IR)

a ieast some homology with various trammembrane receptors, such as the PDGF receptor

(kil, see ref 35) and the insulin receptor (ros and met, see ref. 36). Several other oncogene

products do not express a membrane spanning domain but becorne associated with the

inner side of the plasma membrane as they are myristylated (37). This group comprises

the products of the mc, 1161, andfis oncogene families. They exhibit a tyrosine-specific

kinase activity, similar to that of PDGF, EGF, or the insulin receptors (38).

Receptor activation through ligand binding altas the conformation of the "signai

particle" presenting an active kinase domain for phosphory1ation of specific substrates.

The phosphorylation of protein substrates located at the ceii membrane a d o r cytoplasm

on tyrosine triggers pst-receptor signal transduction d e s (39). Several proteins are

known to be phosphorylated through growth factor-associateci kinase activity and are

believed to be do- elements of receptor-associateci signal transduction pathways.

They include phospholipase Ç (PLC) (40). GTPase activating protein (GAP) (41),

phosphatidylinositol 3-kinase (PL3 kinase) (42). and the c-raf protooncogene product

(43). Phosphorylation of P L Ç results in the generation of inositol 1,4,5-triphosphate

(IP3) and lY2 diacylglycerol (DAO). In hun, DAG and ïP3-induced calcium release cm

stimulate protein lcinase C (PKC), a serine threonine kinase (39). PKC is one of the best

characterized in a series of protein-modifying enzymes which are thought to interact in

the integration and transmission of regulatoly stimuli which ultimatey influence ceIl

cornmitment to proliferaton or diffkrcntiation. The most immediately evident of receptor

signal transduction is the transcriptional activation of early response genes. Among these

Li Long. Regrchtion of Tm01 Cell I&on and Metasftwis by the Typc I IruuIui-liùe Growth Factor Reccpror (IGF-IR)

are at least hvo protooncogenes - c-fos, which itself encodes a transcription factor (44)- is

activated within 5 minutes of administering mitogens to quiescent 3T3 cells , while c-

myc, the product of which is r e q u i . for progression to DNA replication (45)- is

transcribed somewhat Iater (one to three hours, see tef. 46,47).

Growth factors may also influence celi growth by affecting the structure and composition

of the extracellular matrk and thus disturbing or altering the interaction of ceus with their

growth substratum. A numkr of growth mrs *ch can affect the proteolytic balance

of ceiis have ken identifid and they may play a role in pmmoting turnor invasion (48).

For example, EGF and TGFa stimulate the secretion of both iaokinase-type and tissue-

type plasminogen activators (u-PA and t-PA) (49) which can convert the inactive

plasminogen to plasmin - a widespectnrm proteinase. Basic FGF is also an effective

stimulator of u-PA synthesis and secretion (50).

On the other hanci, proteinases can contribute to the processing of growth factors thereby

regulating their bio-avdability. For example, plasmin was teported to convert the high

molecuiar weight precursor of TGFQ into the mature TGFB dimmer (51) while type IV

collagenases whose expression can be regulated by EGF were found to degrade the IGF

binding proteins produced by rat placenta and mouse osteoblast cells (52, 53) thereby

modulating IGF-1 hc t ion . Thus a reciprocal relatioaship exists between growth factor

production and proteinase synthesis and activation. The d e of the iasulin-like growth

factor I in malignancy which is the focus of this shdy is reviewed in greater detaii in

Li Long. Rc&uion of Timor Cell i m i o n and h&mtarü flic TF f fiuuIUI-Iikc Gmwth Factor Recrpior (IGF-IR)

a Chapter Ii. A summary of the polypeptide growth factors and thek properties is provided

in Table 1-1. In Table 1-2, the evidence for p w t h factor and growth factor receptor

involvement in malignant progression of various human tumors is summerized.

*References for Table 1-1: PDGF: ni. u. sq; EGF & TGFa: (5s. sq; TGFP: ( 5 3 ; IGF: (5s. m;

FGF: (60,611; MGSA: (62); IL-3: (63); M-CSF: (63); G-CSF: (63); GM-CSF: (63)

Table 1-1. Properties of Polypeptide Growth Factors implicated in malignancy

Known Sources Known Targets Receptors I Rcfenncfs Factors PDGFaa, ab Dimen of A (17 kDo) and B (16 k h ) 1 mdW chains. B chnin is produci of c-sis proie 1

-m

Two spcckti of glyqmceins. hah iyrosiiw Idnws. T y p a (170 kDa) binds dl PûGF dimus. Type B ( 180 kûn) binds PDGF bb

- . .

Plaielis, plnœnta, preiinplantation ernbryos, EDdMhclial cells

Mcscnchymal , glial end s d mwk cclls.

and ab V d d y Roccin tyrosine kinase (175 kDa). Roûuct of SS,56 Mnjor f m -6 kDa. Some larger spccics

TOP-a daecced. EGP and TGPa pcaeins am 4û% identicai. Boih r e W by pocadysis of membrane-bounâ

EGF: suômaxillary g l d , Brunncrs gland, a

mRNA (but no 6 kDa paiein) in vprieiy of newbom mouss b u s , TG-: Rcimpiantiiion m m cmbryos, her embryos, plaœnte Common in iruisfomiod œU1.

Epiihel id, mc~nchymal, and dirl œlls tb c c M prolo-omcogcne. Wc4xpcor for

EGP, TC3FJ-q and vwcinia vinir gmwth factor.

Typa 1 S@W kûa, type 2 1 15-140 kDa, type 3 280-330 &Da, Each typa binds TOPPI, -

snd -83, Type 1 nuy ôa main mdioior of ICIpoorcr. lGPl mmpor (130 kDa t 90 W8)1 proCCin tyrosim k b c , Mn& IGPl d 4. IGP-II rtcepoi (250 kDa) binâs IGP-Il, idcniicd IO

muuiofie.o.phos~ m p o r .

Wide vuiecy oîœll typer

I 1

lGP-l nd Il 1 7 kûa High homology tocach oihtr and 1 lGPl d n l y podwed in liver. IGPll mRNA in variety of celis, includhg sane tumor ah, kii patin r#naimer undcc6asbb. Bah pn#al in p h in associahm wiih rpscihc ôindinp pcoreins. Low mRNA hvtlr in wida mga of nomvl and msfdal , nadnr W l y dltnbured, associwcd wiib erinallular mslrlr.

I to proinsulin.

Melanom cclls, mRNA dcrecUMa in oWn.

140 kDi W e i n tyrosine kinase 63 150 kDa poiein tyrosine k i m . Roduct oî 63 ibs cfm pcoi0-011C~na monomus, Alremaiive pmducîs of

âiflcrcntirl splking. O-CSP 24 kDa glycopracin Macrophages, fibobh, endothclid cells,

OM-CSP 14 kDa glycopmtcin

Abbreviriions: PDGF, plnickt-denved gmwîh frclor; EGP, cpidrrmal growîh fWor; TGP, iransfomilng growth factw; KIF, fiboblrrr growih faclor; IGF, insulin-like growih firior; MGSA, ri~lrvioiiiu growth-riimiloiing nctivity; IL, iwrkukin; M., G-, and GM-CSP, --, granulocyte-, ud ~iocyidmecfOphPec-colcmy dmulsiing factor, This t g k incqoraes d i e s piblishtd by &r nuthon anâ dcmiùcd in the mcntioncd rcfcr#içef

Li Long. Replation of Twor Cd Ihasion and ACktcut4iu by rlir Typr Ilmiin-lik Growth Factor Roaptor (IGf-IR)

Table 1-2. Detection of Growth Factors and Growth Factor Rmptors in Various Stages of m.lignant progression

Tumor Type L u g Breast

S tomach

Liver Endometriu rn Kidney

Bladder Prostate

GF; Assay Normal Benign GFR

EGFR ICA IGF-II RNA + - * IGF-IR Protein - + TGF-a RIA + PDGF mRNA + FGFs mRNA + IGF-LI mRNA + p@ ICA + +

EGFR ICA + EGFR DNA + bFGF 1 mRNA 1 - 1 +

Ref.

Abbreviations: EGFR, epidennal growth factor receptor, E, estrogen; ER, estrogen receptor; IGF-II, insuüa-like growth factor II; TGFu, transforming growth factor a; PDGF, platelet-derived p w t h factor, FGFs, fibroblast growth Eictors; PgFt, progesterone receptor; TGF-P, transforming p w t h faftor P; bFGF, basic fibroblast growth factor, RIA, radioimmunochemicai assay; ICA, immimocytochemicai assay.

1.3 The Wace-ix - DagtadjllQ Protainaaaa

During the development of invasive tumors, tumor cek de@ the "social order" of organ

boundaries and cross into 'Toreign" tissues. The mammalim organism is divided into a

series of tissue compartments separated by the extracellular matrices consisting of

basement membranes and the interstitial stroma (6,72). During the transition kom in situ

to invasive carcinoma, tumor celis penetrate the epithelial basement membrane and enter

the underlying interstitial stroma to interact with the stromd cells. Thus, one definition of

the behavior of the metastatic tumor cell is the tendency to cross tissue cornpartment

boundaries and intermix with different celi types (6,73).

The contiuuous bascment membrane is a dense meshwork of collagen, glycoproteins, and

proteoglycans which does not n o r d y permit the passive passage of ceiis (72). It may

ais0 be a storage depot for latent proteinases and cytokines including angiogenesis

factors, which can be activated or released during processes such as wound heaLing and

aiso by mediators associated with invading celi pseudopodia (74). Once the tumor ceils

enter the stroma, they gain access to lymphatics and blood vessels for M e r

dissemination. The interaction of tumor cells with the ECM can be divided into three

steps namely, attachment, matruc dissolution, and migration. The nrst step in this process

is adhesion of the tumor ceii to basexnent membrane proteins. This adhesion is mediated

mainly by cell surface receptors of the integrin family (75, 76) and also by non-integrin

receptors such as the 61 kDa laminin receptor (77, 78). These receptors recognize

collagen as well as ECM glycoproteins such as laminin, vitronectin and fibmnectin.

Following adhesion, a locaiized zone of lysis is produced in the basement membrane at

the point of tumor cd-ECM contact (7). h viva -or cells can produce ECM degrading

enzymes (79) or îhey can induce host cells such as stroma1 cells and innltrating

Li Long. Regirlrrtion of Tmor Ce11 Inwuion and .Werawsis by the Typc I InsuIin-likc Growih Factor Recepror (IGF-I R)

a leukocytes to elaborate the proteinases (80). ECM Lysis generally occurs in a highly

localized fashion, in regions of celi-ECM contact (81), where the balance of active

proteinases and natural proteinase inhibitors has been dismpted resulting in excess

proteinase activity. Locomotion which propels the tumor cell across the basement

membrane and through stroma is the third step of invasion . It is now recognized that

random tumor ceIi motiiity can be regulated by tumor cell cytokines such as autocrine

motility factors (82) and scatter factors (83). In addition, the direction and site of tumor

ce11 locomotion may k inf luaid by host organderived chem-ts such as IGF-1

(84). Such chemoattractants could play a d e in organ-selective homing of metastatic

cells. This could wmplement 0 t h mechanisms of organ homing which include

preferential adhesion to organ-specific endothelium (see below) and preferential growth

in selected organs due to local growth factors (85,86).

The matrix degrading proteinases, which have been implicated in malignancy, can be

grouped into at least five classes on the basis of their active site, requirements for optimal

activity such as pH and cations and susceptibility to specifïc inhibitors (Table 1-3). The

evidence for their role in invasion and metastasis has been comprehensively reviewed

elsewhere (87)

Serine protehases

The serine proteinases most extensively investigated in the context of rnalignancy are the

plasminogen activators (PAS). These enzymes couvert plasminogen (Pg) to its

Li Long. Regdation of Tunuw Gd Invasion and Mcl~frasris by rhe Typc I Insulin-lik Gmwrh Factor Recepror (IGF-1 R)

Table 1-3. Major Claues of Matrix Degrading Enzymes and Inbibitors Implicated

Class of Members inhibitors Inhibitors References Proteinases (Natural) (Experirnental) Senne uP A PAI-1, PAI-2, PN-1 DFP, PMSF, TLCK, (88)

Zn". leupeptine, uanexamic acid, -dine

tPA PAL 1, a2-antiplasmia Idem (89) Pla~min PM-2, PN-1, a2- AproÉnin, SBTI, (90)

(91) antiplasmin EACA (92)

LEI TLNP Elas tinol Cathepsin G

Cysteine or Cathepsin B (-likc) Cys min, antipain, Iodoacetate, N-ethyl- (93) thiol MAF, ala2TPis rnaleimide, E64,

TLCK, 4- chIorornercuninzoa (94)

Cathepsin L Idem te, leupeptin

a Idem Carboxyl or Cathepsin D (-like) Pepstatin Diazoketones (95)

asparac Metallo- or MMP- 1 or MMP-8 TTMP EDTA, EGTA, Dm, (96)

Zinc 1.10-pheaantroiine (97) (98) MMP-2 Idem Idem, SC-44463 (99) MMP-9 Idem Idem (100) Collagenase V Idem Idem (101)

MMP-3 Idem Idem (1W MMP-IO Idem Idem (102)

MMP-7 Idem Idem m-EL Idem

Glycosidase P-N-Acetyl- (87) glucosaminidase Heparanase (87) Hvaluronidase (103)

Abbreviations: uPA: urinary type of plasrninogen activator, @A: tissue type of plasminogen activatot; 1-El: leukocyte-type elastase; TLNP: trypsin-like neutrat protease; MMP: matrix mctalloproteinase; MMP-1 or MMP-8: = interstitial collagenasc a vcrtcbnte collagenasc; MMP-2: 72 kDa type IV collagenasc = gelatinase-1; MMP-9: 92 kDa type IV collagenase = gelatinasc-2; MMP-3: strornclysin-1 = transin-1 = proteog 1 ycanase; M W - 10: sirornelysin-2 = transin-2. DFP: diisopropyl fluorophosphate; PMSF: phenyimethylsulfonyl fluoride; TLCK: Na-p-tosyl-L-lysine chloromethyl ketone; PM-1: endothelia1 type of pbminogen activator inhibitor or type 1; PAI-2: placental

interstitial colIagenase = vertebrate collagenase; MMP-2: 72 kDa type IV collagenase = gelathase-l; MMP-9: 92 kDa type IV collagenase = gelatllwe-2; MMP-3: stromelysin-1 = tramin-1 = pro teoglycanasc; MMP- 1 O: stromelysin-2 = transin-2. D FP: di isopropy 1 fluorophosphatc; PMSF: phen ylmethy lsulfony 1 fluoride; TLCK: Na-ptosy 1-L-lysine chlorornethyl ketonc; PAI-1: endothelial cypc of pIasminogcn activator inhibitor or type 1; PAI-2: placental type of plasminogen activaîor ïnhiiitor or type 2; PN-1: pro- nexin 1; SBTI- soybean üypsin inhibitor; EACA: E-aminocaproic acid; MAF: inhibitor human amniotic fluid; aldTPIs: al-, a2-thiol protease inhibitors; €64: L-trans-epoxysuccmyi-Icucylamido (4guanidino) butane; TIMP: tissue inhibitor of rnetalloproteinase; EPA: erythtoid potentiating -or; EDTA: ethylcnc-dîaminctetetraacetic acid; EGTA: ethylene glycol-bis (paminoethyl ether) N,N,NT,N'-tetraacctic acid; DIT: DL-dithiothrcitol; SC-44463: Searle cornpound 44463.

This table incorporates studies published by other authors and descriid in the relevant references-

and Val-561 peptide bond (88). Two different PAS have been identified in human cells

isolated fiom urine, hence its name (104). When fkst released h m by the celis, uPA is a

single-54 kDa-chain (105). First observed to be a zymogen in kldney ceil cultures and

termed prourokinacp!, this single chiiin uPA is converteci through proteolytic digestion by

plasmin or m i n at the Lys458 - Ile-159 peptide bond (106). This separates the A

and B chains producing a two-chained uPA held together by a disuIfide bridge (107). The

tPA is a 70 kDa glycoprotein produced under physiologic conditions primarily by

endothelial cells. It can be found in the plasma at a concentration of 5 ng/ml, mainly

bound to its inhibitor, PAI-1. It is rapidly cleared by liver d s , with a plauna ha-Me of

about 5 minutes (108). The plasminogen activaton are products of distinct genes and may

be produced by the same or distinct cells (88). The enzymatic activities of the PAS can be

inhibited by specinc cell-denved inhibitors of plasmiwgen activation (PAIS) including

PAI-1, PAI-2 and PN-1 (protease nexin 1) (109). Through the activity of PAIS, ECM

proteolysis is tightly regulated. Plasminogen is a ubiquitously distributed glycoprotein of

0 about 92 kDa produced in the liver and found in the semm at a concentration of 1-2pM

0 with a biologic half-life of 2.2 days. Plasmin has a broad degradative activity on ECM

substrates as well as an abiiity to activate zymogens particdarly procollagenases (1 10).

Another serine pmteinase implicated in malignancy is the Ieukocyte elastase (90). This

protease shows broad substrate specificity as it degrades, in addition to the highiy

insoluble elastin, severai other ECM molecules such as types III and N coliagen,

fibronectins and proteoglycans (1 1 1).

O

The Cathepsins

The cathepsins are a biochemically heterogeneous group of lysosorna1 proteases with

broad substrate specificity. They include thiol and carboxyl proteases, most of which

have optimal activity at acidic pH. Cathepsin G is a serine protease with optimal activity

at neutral pH and is found in the azurophi1 granules of neutrophils and monocytes (92). in

normal celis, cathepsin B, a cyrtine p r o t e k , is routed as an active 27 kDa form into the

lysosomes. However, in tumor cek cathepsin B activity was found in association with

plasma membrane fiactions and in shed membrane vesicles (93). Plasma membrane-

associated cathepsin B could not elute easily by various treatments, including maonose-6-

phosphate addition. A secreted high molecular weight cathepsin B form with Limited

proteolytic activity, i.e. the 43 kDa proenzyme, has been identified in culture media of

human breast carcinoma, mouse carcinoma and melanoma ceUs and rabbit carcinoma

cells (1 12). These secreted fonns can be M e r processed and activated extracellularly

either by limitecl pepsin digestion or by autoactivation at pH 3.0. Tumor-denved

Li Long. Rcgu farion of T.untof C d I . i m and LHC~CLSIQIU by rkr T m I Innrfin-Iik Growth Facror Recrpror ( IGf- IR)

cathepsin B have also been shown to have activity at neutral pH (1 13). In various tumors,

cathepsin B was detected at the invasive fiont and its expression at the ce11 surface or

secretion which are rarely observeci in normai cells were shown to correlate with the

invasive/metastatic phenotypes in several tumor celi lines (114). Another cysteine

protease which is normally targeted to the lysosomes where it is proteolytically activated

at acidic pH is catbepsin L (94). The precursor fomi of cathepsin L was identified as the

major excreted protein (MW) of ras-oncogene-transfomeci murine fibrobtasts (1 15).

This form shows limited activity at neutrai pH and becornes m e r activated

autocatalytically at a pH lower than 5 (1 16). Both the synthesis and secretion of MEP

were shown to be dramatidy i n d in tesponse to tumor promoters, growth factors

and viral transformation (1 17). In a study by Maciewicz et al, huxnan invasive colorectal

carcinoma celi lines were reporteci to secrete mature and active f o m of cathepsin L,

whereas adenorna-derived ce11 lines secmted inactive precursor forms of the enzyme

(1 18). Another cathepsin implicated in rnalipmcy particularly in breast cancer is the

carboxyl protease cathepsin D. Synthesis and extracellular secretion of cathepsin D are

inducible by estmgen and growth factors both in primary cultures and Wim (95).

Cathepsin D is semeteci as an inactive 52 kDa precursor form and autocatalytically

processed into a 5 1 kDa fom which is active at acidic pH (1 19). Cathepsin D, secreted by

breast cancer cell and autoactivated at pH 4.5, was reported to degrade p a the

subendothelid extracellular matrix (1 20).

The matri. metailoproteinma

The rnatrix metdoproteinases (MMPs) can be broadly divided into three major groups

on the basis of biochemical properties and structurai organization (121): They are the

interstitial vertebrate coilagauixs which degrade types 1, II and iIï collagens (MMP-1

and MMP-8); type IV collagenases or gelatinases (MMP-2 and MMP-9); and the

stromelysins ( a h known as proteogiycanases, transius and MMP-3, MMP- IO and MMP-

7). MetalIoproteinases in g e n d hction at n e d pH, require Zn2+ (and ca2> ions as

cofactors and can be inhibited by chelators such as EDTA, EGTA and by synthetic

inhibitors such as DIT or 1,lû-phenanthdine. Their activity is reguiated by naturai

inhibitors known collectively as tissue inhibitors of metalloproteinases (TïMPs) (122).

Al1 members of this family share several weli-co~lsecved domains, including an amino-

terminal domain in the mature active molecules, a fibmnectin-iike coilagen binding

a domain (in type N collage-), a central 2n2+ -binding domain carrying the active site

and a carboxy-terminal hemopexin-like domain (123). These enzymes are secreted in a

latent zymogen form following cleavage of a signal peptide. The naturai activators of

these enzymes Ur vivo are still a matter of active investigation. vitra , efficient

extracellular activation of the m e t a l l o p m t e ~ can be achieved by treatment with

organomercurïals such as m d y l , phenyimercuric acid and hophenyImercuric acetate

or APMA (124). This activation triggers autocataiytic cleavage resulting in the removal

of a propeptited N-terminaiiy to the conserved PRCGVPDV sequeme and a cysteine-zinc

bond is disrupted. This dissociation of the cysteine residue fkom the zinc atom and its

replacement by water is believed to expose the active site and convert the enzyme to a

catalytically active form, a process m e d the "cysteine switch" mechanism (125).

The net proteolytic activity mediated by the metalloproteinases can be regulated at

several Levels: at the tmscriptional level, at the level of translation, secretion, proenzyme

activation and proteolytic activity. TIMPs are ubiquitous and potent natural inhibitors of

the metalloproteinases (124). The best studied are TIMP-1 and TIMP-2. Both can form a

complex at 1: 1 stoichiometry with either activated metalloproteinases or the proenzyme

(124). Tt is of interest to note thaî the same microenvironmental factors which cm

modulate PA activity namely growth fhctors (e.g., EGF, TGF-a, PDGF, bFGF),

cytokines (e.g., IL- 1, TGF-p), stemid hormones (e.g., glucocortiwids, sex hormones),

ECM components (e.g., coilagen, fibmnectin, laminin), and naturai (e.g., retinoids,

lipopolysaccharides, eicosapentaenoic acid) or synthetic compounds (e.g., phorbol esters)

can also modulate net mdoprotease activity (126).

Studies using synthetic metalloproteinase inhibitors or genetic alteration in TIMP

production provided compelling evidence that metallopmteinase activity is indispensable

for the invasive/metastatic phenotypes. A m k e d reduction of TIMP production by

rnalignant vs. benign murine fibmblastic tumors and increased coiiagenolytic activity in

various mesenchymal and epithelial &ors have been reported by various groups (127,

228). The consistent inhibition of invasion &I v i t r ~ by 1,lO-phenanthroline or by natural

or recombinant TIMP provided striking evidence for their role (129). In addition the peri-

and intratumorai degradation of fibrillar collageos, which is regularly observeci in human

biopsies, impücate interstitial collagenase (MMP-1 or MMP-8) in tumor invasion and

Li Long. ReguL~ion of Tumor C d I'ion and ~Uefasfasu by r k 7ipc 1 ImIin-Iike Growth Factor Recepror (IGF- I R )

metastasis. The role of type IV coilagenases in invasion and metastasis will be discwed

in greater detaii in Chapter III.

Glycosidrrses

Glycosidases have also been implicated in malignancy. Abmant glycosylation has been

dernonstrated in malignant ceils (130) and altered glycosylation may influence both celi-

cell and celi-substrate interactions at various stages of the metastatic process. Tumor

lysosomal exocytosis has ken proposcd as one possible mechaniSm responsible for the

extracellular expression of P-N-acetyigiucosaminidase and endo-~glucuronidase (87).

An hcrease in the BON-acetylglucosaminidase levels was obsetved concomitantly with

the appearance of spontaneous (micro-)metastases of subcutaneously inoculated Lewis

a lung carcinoma ceiis (1 3 1). Similar fïndings were obtained with B 16 melanoma variants

and human carcinoma cells (132). ïa addition, glycosiks , in particular

endoglycosidases, rnay play an important d e in directional cell motility by as they may

contribute to local degradation of ECM. Sulfateci glycosaminoglycans and proteoglyuuis

can be considered ceii-immobWg molecules because they stabilize cell adhesion (1 33).

Indeed, proteoglycans such as heparin sulfates, were shown to be more susceptible to

degradation by d g n a n t cells (134) and a rat carcinoma was reportedly shown to secrete

a hyaluronidase (1 03). Endoglycosidases are synthesized by several normal ce11 types,

including fibroblasts, platelets and inilammatory macrophages (135). However, endo-p-

D-glucoronidase (hepamme) is preferentially expressed by highiy invasive and

metastatic tumor cells as compared to l e s dignant variants or normal cells. For

Li Long. RegirIation of T m w Cell Invaion anâ lUetasrasis by the Tjpe I Innfin-Iike Growth Factor Recepror (IGF-IR)

a example, increased expression ancilor activity of heparanase was found in a highly

invasive andor metastatic mouse B l6 melanoma variant (1 36), a mouse Eb T lymphoma

variant (137), rat rhabdomyosarcoma ceii variants (138) and a mouse fibrosarcoma (139).

The enzyme found in human and murine melanoma cell lines is a 96-kDa ceil-associated

protein with optimal activity at pH 5.6 but signifiant activity also at a physiological pH

(1 39). Specific inhibition of this hepiaanase by chemicaily modified heparias reduced

ECM-degradative and lung colonization potential of B 1 6-BL6 melanoma (1 40).

Treatment of rat mammgFl carcinoma ceUs with d a t e d polysaccharides likewise

resulted in the inhibition of a tumor ce11-derived heparanase, in decreased ECM

degradation and in decreased metastasis (141).

1.4 The role of cell adheaionmaleculeo

Cell-cell and ceii-matrix adhesion are of paramount importance in embryogenesis,

morphogenesis, idammatory responses, hemostasis, and maintenance of tissue integrity.

Quantitative andior qualitative changes in cell adhesion have b e n demonstrated in a wide

variety of pathological conditions including neuromuscular and neurological disorden,

chmnic inflammation, as well as in -or progression and metastasis (142-149). The

molecules mediating ceiiular adhesion have been grouped into several distinct families,

the most prominent of which are the integrins, rnembers of the imrnunoglobulin (Igs)

supergene family, cadherins and selectins. A large number of other adhesion molecules

which de@ categorization into any of these f d e s have been described and they include

cell surface proteoglycans such as CD44 (150, 151), glycopmteins (152).

a glycosphingoiipids (153) and several laminin receptors (154) some of these molecules

have also been implicated in metastasis.

-: The integrios are heterodimeric trammembrane giycoproteins consisting of an

a subunit noncovalently associated with a B subunit. To date, 11 B subunits and 15 a

subunits have been teported (155). These subunits can combine to form more than 20

distinct integrin heterodimers (155-157). Most integrins bind to extracellular matrix

proteins and promote ceU-substrafum adhcsion. However, some integrïns recognize

integral membrane proteins of the irnrnunogiobulin supcrfamiiy on other ceils and

mediate cell-ceii adhesion (158). Both the a and the $ subunits of integrins are membrane

glycoproteins with a large exîraceliular domah, a single membrane spanning segment

a and a short cytoplasmic portion. The ligand-binding pocket of the integrins consists of N-

terminal peptides of both subunits. Divalent cations, such as ca*, M~~ and MU* are

required for the association of the a and subunit and for ligand binding (159, 160).

While the extracellular portion of integrins binds to an extracellular ligand, the

cytoplasmic domains intetact with cytoskeletal elements (161). The integrins can transmit

signais fiom the extracellular matrix to the ceil interior (outside-in signaiing) (162). They

are also a target of regdatory signals originating h m the ce11 interior (inside-out

signaling) (163). The interaction of integrins with cytoskeletal elements leads to the

formation of specialized adhesive jimctions, such as focai adhesioas and

hemidesmosomes (164). In accordance with the e d y £Ming that focal adhesions contain

elevated levels- of phosphotyrosine (165), recent observations implicated tyrosine kinases

0 such as focai adhesion Linase (FAK) (166) and c n k (167), which localize in focal

adhesions, in mediating integrin signaling. Both FAK and c-crk also appear to be

involved in the signal transduction by growth factor ceceptors (168) indicating that they

may potentially integrate the signals originating h m integrias with those elicited by

growth factor receptors. In addition, adhesion of various ceU types to fibronectin and

other ligands also causes elevation of cytoplasrnic pH (169) and calcium influx (1 70),

suggesting that integrins may also activate the phospbatidyl-inositol polyphosphate and

protein kinase C pathways.

The role of integrin receptors in invasiodmetastasis cm Vary, depending on the receptor

subtypes and the histological type, grade, and stage of the cancerous lesion (155). In

0 general, transformed and malignant cells express reduced level of the integrin (171-

173) which may explain the general reduction noted in the adhesion of maügriant tumor

cells to fibronectin and extracellular matri.. In contrast, some carcinomas were shown to

express increased levels of the integrin laminin receptors a6P, or a6P4 (1 74-1 78) and this

rnay regulate their metastatidivasive ability. On the otbet hand, expression of many

other integrins such as a2P,, and a3P1 may e i t k remain unaltered or increase in some

tumor types but decrease in others (171,176-178). The vitroaectin receptor has been

irnplicated in the migration on vitronectin of severai diffèrent celi types including

endothelid cells (179), macrophages (180), neural crest cells (1 8 l), smooth muscle cells

(182) and malignant ceils such as lung and p a n M c carcinoma (183) and melanoma

cells (1 84). Vitronectin receptor-mediated migration on vitronectin is an important event

Li Long. Reguiacion of T h o r Celf invasion and lHutas~asis by tk Tjqu 1 Insufin-like Growrh Factor Receptor fIGF=I R)

a in physiological processes such as angiogenesis (185), wound healing (186), and

embryonic development ( 18 1). Vascular cells expressing the receptor can migrate to form

new blood vessels, a pmcess required for metastases formation, Tumor ceiis, including

malignant melanoma cells produce angiogenic factors such as bFGF which trïgger this

process (187). integrin a&-mediateci adhesion to vitronectin also appear to rescue

melanoma cells fiom apoptosis (187). A recent study in our labonitory revealed that the

expression of the uroliaase plasminogen activator receptor (uPAR) in metastatic

melanoma celis is linLcd to the expression 4 h c t i o n of the integrin vitronecth

receptor (188, 189). A positive correlation was also demonstrated between

expression of integrin a&, and the metastatic potential of melanoma cells (1 90, 19 1).

0 be C-: The cadherins are family of ca2+-dependent transmembrane

glycoproteins that mediate mainly homophilic but also heterophiiic cei.i«ii adhesion.

Three major subclasses, Le., E-cadherin (epithelial cadherin; uvomonilin; L-CAM), P-

cadherin (placental cadherin), and N-cadherin (neural cadherin; A-CAM) have initially

been identifieci and are well characterized at the molecdar level (146, 147). Ln recent

years, many new cadhcrins or cadherin-iiice molecules have been identifid including the

V-cadherias expressed on endothelid celis, R-cadherin in retina, B-cadherin in brain, M-

cadherin in muscle, T-cadherin that bas not been clearly defined, cadherins 4-1 1 found in

the nervous system, and desmosomal cadherins (146,147,155).

a The structure of a typical cadherin consists of an amino-terminal extemal domain having

five tandem repeats, a single trammembrane segment, and a cytoplasmic carboxy-

terminal domain of about 150 amino acids (192). The binding hinctiom of the cadherin

are localized in the amino-terminal tandem repeat, M e the other repeats contain

putative calcium binding sites (193). The cytoplasmic domain of caâherins interact

strongly with a group of intracellular proteins known as catenins and with plakoglobin

(194). The catenins are thought to mediate the interaction between the cadherins and the

cytoskeletal microfïlaments. Ranadrably, the cadherins cannot promote ceil adhesion

unless they are complexed with the catenins (195).

Similarly to integrins, cadherins are also Liaked intraceilularly either to microfilaments

a (for classic cadherins) via the a and f3 catenins, or to intermediate filaments (for

desmosomal cadherins) k u g h pldcogiobin and desnoplakllis. Cadherins utiiize the tri-

peptide His-Ala-Val present within the first extracellular domain as the recognition

sequence to initiate homophilic c d - c d interaction (146, 147). The regdated expression

of cadherins plays an important role in ceU-sorting, controlling ceil polarity, and

regulating morphogenesis.

An inverse correlation has ken noted between E -cadhe~ expression and the

invasivdmetastatic potential of animal and human tumors (155). Dom-regulated E-

cadherin expression is probably one mechaaisms responsible for the loss of cell-cell

contact, an initiating step in tumor progression and generation of metastatic variants. An

Li Long. Regulation of T m o r Cell Inwrion and ,Hetas~is by rlrr Tjpr I Insufin-likr Growth Factor Recepror ( K F - t R )

inverse correlation between E-cadherin expression and the degree of differentiation has

been reported in many carcinomas (196, 197). Transfection of E-cadherin cDNA into

epithelial tumor cells could restrict or reverse the invasive behavior (1 98, 1 99). Decreased

E-cadherin expression was recently correlated with increased grade of human prostatic

cancer and with poor prognosis in prostate cancer patients (200,201). The reduction of a-

and pcatenin expression, as weli as that of E-cadherin, was significantly associated with

tumor dediffitiation, Mtrative growth, and lymph no& metastasis. In squamous al1

cancer of the esophagus, the tumors expressing E-cadherin but with selectively reduced

expression of a-catenin did not show tight cell-cell adhesions and metastasized to lymph

nodes more fkquently than tumors expressing both E-caâherin and a-catenin molecules

(202). Moreover, the Spearman rauk correlation coefficient (rs) of a-catenin expression

. with differentiation or lyrnph nde metastasis was higher than that of E-cadherin in

human esophageal and breast cancers (202, 203) . These results indicated that the

reduction of a-catenin expression is more signincantly correlated with the invasive

phenotype and with lymph node metastasis wmpared with that of E-cadherin expression.

hterestingly, fells expressing mutant cadherhs that lack p-catenin binding sites were

shown to adhere to each other more tightly than those with normal cadherins (204).

0: Members of the Ig supergene family

(IgSF) are cell SUTface adhesion molecules which possess immunoglobulin-like folds in

the extracellular domain(s). Most members of this famiiy mediate ca2+-independent

adhesion, but some mediate ca2+-dependent adhesion processes (172, 205). They can be

Li Long. RegutOtton of Tmor CeU fnvarion a d Afetatrasu by the Typr I I w I i n - l i k e Growth Factor RPceptor (IGF-f R)

arbitrarily divided into several subgroups. These include immune ce11 receptors such as

the T ce11 receptor, Ig !I and L chah, CM, and CD8 (206), neuronal N-CAM, Ng-CAM

and Nr-CAM which ~ IE prototype of ce11 adhesion receptoa in this family playing role in

embryogenesis and morphogenesis (147, 207), growth factor receptor such as the PDGF

and CSFl receptors (172, 205), platelet and endotheîial ceil receptors such as PECAM-

lKD3 1, ICAM-1, and VCAM-1 (208, 209) which play a role in inflammation and

angiogenesis and members of the CEA (carcinoembryonic antigen) family (210, 21 1)

which are nonnally expressed during embryogenesis and are upregulated in some

malignant cells. Members of IgSF can be uivolved in homophilic adhesion (e.g. N-CAM

- N-CAM interactions) or in heterophilïc adhesion such as VCAM-1 binding to integrin

a43, and ICAM- 1 binding to integrins LFA- 1 and Mac- 1 . CEA and DCC (Deleted in

a Colon Carcinoma) appear to fiinction as dominant and recessive metastasis-relateci

oncogenes, respectively (212). CE& a widely useci human tumor marker, was shown to

mediate ca2+-independent, homotypic aggregation of human colon carcinoma cells and

colon carcinoma celi adhesion to collagen m M and was also locaiized to cell-ceii

contact sites in situ (2 10, 2 1 1). A direct positive correlation has also k e n observed

between serum CEA levels and the aggressiveness of human colorectal carcinoma cells in

nude mice (213). DCC is a tumor supprrsser gene located on human chromosome 18q

which encodes an NCAM-like adhesion molecule (214). It has k e n postulated that

deletion of this gene (similarly to loss of E-cadherin) resdts in the loss of celi-ce11 contact

(2 13). Another IgSF member, ICAM-1 is expressed on melanoma celis. Levels of ICAM-

Li Long- Rrguia~ion of T'or Ce[[ Imasion ami , W e m m u by the T j I Iitsuiin-like Gmwrk Factor Receptor (IGF-IR)

0 1 have been shown to positively correlate with metastasis and it has been proposed that it

is involved in homotypic ce11 aggregation of melanoma cells (21 1,215).

S e l e c m (du, t e d LEC-CAMs) are adhesion molecules which recognk cell surface

carbohydrate ligands. Stnicturally, all selectins contain an N-terminal lectin domain, an

epidermal growth factor-like module, a variable number of complement-binding repeats,

a transmembrane domain, and a short cytoplasmic tail (155). The selectins mediate

lymphocyte-homing and leukocytt migration and an exprrssad on Ieukocytes, platelets

and endotheliai cek (155). Three members have ken characterized. L-selectin (gp90m'4;

MEL14; LAM-1; LECAM-1) is expresscd on neutrophils, monocytes and lymphocytes.

This selectin, together with CD44 and integrin a& , mediates lymphocyte homing to

iymph nodes and neutrophil adhesion to the EC at sites of infhmmation (216). E-selectin

(also known as ELAM-1) is expressed on vascular EC stimulateci with IL-1, TNF-a or

endotoxin, and mediates targeted adhesion of neutropbiis and monocytes at sites of

inflammation (2 16,2 17). E-selectin mediates leukocyte rolling, the first step in a cascade

of leukocyte-EC interactions 1-g to tramendothelia1 migration (218). It also acts as a

specific homing receptor for a skin-associated memory T cell subset during chronic

inflammation (219). P-seIectin (GMP-140; CD62; PADGEM) is loçalized in the granules

of platelets and in Weibel-Palade bodies of EC. It is constitutively expressed in normal,

noninflamed tissues (2 16, 21 7, 220), but its translocation to the ce11 surface where it can

serve as an adhesion receptor for neutrophils and monocytes requires ce11 activation by

thrombin (platelets), histamine, phorbol esters, or oxygen radids (2 16, 220-222). The

Li Long. Rrguhrion of Tumor Cclf ?hasiun a d MefasIPIL by the T m I ImIin-fike Growth Factor Receptor (?CF-IR)

major h c t i o n of this adhesion molecule is thought to be the rnediation of neutrophils

adhesion to EC during the earIy phase of idammation (1 55). To date there has been no

evidence of selectin expression on tumor c e k However, various solid tumor cells

express abundant selectin Ligands, Le., siaiyl ~ewis ' and ~ewis* (sLea and sLeX) and the

expression of these ligands is positively correlateci with theîr metastatic potential (223,

224). Furthemore, expression of selectins on cytokine-activated vascular endothelial

cells was s h o w to increase tumor-EC adhesion as weli as experimental metastasis (225).

Taken together, the evidence suggests that this f d y of adliesion molecules may play an

important role in cancer metastasis.

Other adhesion molecules which have been implicated in the process of metastasis are

0 glycosyl-transfii, endogenous lectins, and glycosidases (226). In addition, CD44, a

multifiinctional, rnulti-isoform transmembrane hyaluronate receptor present in endothelial

cells, epithelial ceiis, chondrocytes, fibroblasts, and leukocytes has also been implicated

in the process. Many tumor ce11 types have elevated levels of CD44 protein or rnRNA, or

express new or aitered forms of the molecule (150). A variant CD44 gene was isolateci

fkom a metastatic rat carcinoma celi line which was not expressed by nonmetastatic

clones of the same ce11 line (227). Transfection of this variant gene into nonmetastatic

tumor cells code& on them a metastatic phenotype when injected into syngeneic rats.

A monoclonal antibody to this variant CD44 protein could retard metastasis formation by

the transfected ceil line (227). Subsequentiy human tumor cell lines were found to

express a receptor with sequence homology to the rat variant (228). This alternatively

Li Long. Rcgrrhtion of Timor Ce11 Irrvarion a d Mclp~tasstr by the Typr I fmlin-fïk Gmwth Facror Recepror flGF-IR)

a spliced CD44 may facilitate tumor ce11 movement thmugh the matrùc and adhesion to

endothelial cetls (228).

The formation of new blood vessels, or engiogenesis is essentiai for expansion of the

primary tumor IMSS. In addition, new blood vessels penetrating the tumor are fiequent

sites for tumor celi entry into the circulation (7, 229, 230). Vascuiar tumors may persist

as thin asymptomatic lesions, restricted by the levels of oxygen and by limiteci nutrient

diffusion. In contrast, vascularized tumors can expand l o d y and metastasize. n e first

inducers of angiogenesis to be identified were the basic and acidic fibroblast growth

factors @FGF and aFGF, reviewed in (23 1). Both proteins are members of a family of

0 growth factors that are chanictnipd by high afflnity bindiag to heparin; each lacking a

traditionai signal sequence for secretion. Both, however, can be released fiom celis under

certain circutnstances (23 1). A third, s e c d angiogenic factor was first identifïed by its

ability to elicit vascular permeability (232); Subsequently, this factor, terrned vascuiar

permeability factor or vascuiar endothelial gmwth factor (VPFNEGF), was shown to be

a potent inducer of angiogenesis (233). Recently, two relateâ endothelial growth factors,

VEGF-B and VEGF-C, have been identified (234). Al1 three VEGF genes as well as

acidic and basic FGF are widely expressed in normal addt organs of mice and humans,

suggestive of d e s in tissue homeostasis. Both albFGF aad VEGF bind to receptors on

endothelid cells that are transmembrane tyrosine kinases and are involved through a

signal transduction cascade in gene regulation. Expression of the three VEGF receptors,

Li Long. Regufarion of Tumor Ce11 /&asion ami ,4ktasmsu by rAr Typl f InsuiUr-like Gmwh Factor Receptor (IGF- I R )

tlk, Bt-1, and fl t4 (WGFR-1, 2 and 3), is restricted to the endothelial cells (233),

whereas the four FGF receptors, FGFRI-4, are more widely expressed (23 1). Tumors can

induce angiogenesis through the release of these soluble angiogenic factors or by

triggering their release by host cells such as s t r o d celis (229, 235, 236). bFGF and

VEGF are commonly e x p d in a wide variety of human and animal cancers.

Moreover, they can be detected at elevaîed levels in the urine and senim of cancer

patients (237, 238). However, angiogenesis is not a single event, but a cascade of

processes initiated by activation of m i c r o v d a r endothelial ceils. The cornmon feature

of many factors which induce angiogenesis, such as bFGF, is their ability to exert

multiple effects on capillary endothelial ceiis. Ther include induction of ceii motility,

proteolysis, and ceii prolifêration. Endothelid ceiis resting in the vesse1 are nimulated to

degrade the endothelial basement membrane, migrate into the perivascular space, and

form a capiilary sprout (239). Interestingiy, VEGF and bFGF have been shown to

synergize in angiogenesis assays in Y& (240), indicating that they c m serve

complementary fimctions.

In the pro- of angiogenesis, aeother group of factors also plays a important role - the

angiogenesis inhibitors. The £kst clues to the existence of endogenous angiogenesis

inhibitors came with the observations that a interferon (241) and platelet factor-4 (242)

could iahibit endothelial cell chernotaxis and proliferation, respectively. Several other

angiogenesis inhibitors have been recently identined including thrombospondin- 1 (243),

a 16 kDa proladin hgmemt (244), a 29 kDa fibronectin fragment (245) and the recently

Li Long. Regirfation of T'or Ceil favasion and . U e ~ i s by rhe f Imiin-likc Growch Facror Renptor fIGF-IR)

a identified angiostatin, a fragment of plasminogen (246). These inhibitors keep the activity

of angiogenic factors in check and serve to maintain the endothelid celis in alternative

States of quiescence or angiogenesis.

1.6 fa- -

Cellular rnotility is by no means exclusive to disseminating tumor celis, lymphocyte

trafic being an obvious example for migration of n o r d cells which involves active

motility at vesse1 walls and outside the circulation. Chernotactic molecules are the major

locomotiondriving signals. Cytohes (e.g., IL-1, TNFa, 1.N-y), bacterial products (e.g.,

N-formylated peptides) and other inûammatory products (e.g., complement-derived

peptide C5a; ECM degradation products) are well lcnown potent chemoattractants for

leukocytes (247-249). The gowth factors aFGF and bFGF are major chemoattractants for

endothelid celis and their production is antagonized by TGF-$ (250). On the other hanci,

TGF-P as weli as EGF can stimulate keratinocyte motiiity by inducing fibronectin and

thrombo&ndin synthesis which can in tum trigger motility ; iFN-y antagonizes

keratinocyte motility by inhibiting production of the ECM molecules and thereby motility

(251). A variety of molecules act as chemoattractants for fibroblasts including PDGF

(252), TGF-P (253), fibmnectin (254), and autocrine motility factor (82, 255). For most

of these factors specific cellular receptors have been identified which regulate the

migratory process. Some chemoattractants under restricted conditions can also elicit other

responses, including proliferation, secretion of hydrolases, release of arachidonic acid and

metabolites, incrcased pinocytosis, and increased cell-cell adhesion (256-258). Different

tumor cells can respond to the same chemoattractants (259). Primary tumon generaliy

trigger an inflammatory response resulting in an increased local concentration of

infiammatory chemoattractants which can then mobilue the tumor ceils. Injured vascular

walls are preferentiai sites for extravasaion by tumor cells and this is partialiy due to

local generation of infiammatory chemoattractants (260). Growth factors may also be

potent chemoattmctants for various ceils. Because many tumor celis elaborate gmwth

factors such as TGFs, PDGF and produts of the FGF-Like hst, uit2 and FGF-5

oncogenes, or ovetexpress growth factor rcceptors, it is conceivable that these fkctors aiso

stimuiate motility in an autocrine fashion (82). IGF-1 has been identifieci as a motility

factor for both normal and malignant ceiis (261). We also fouad that IGF-I is a

chemoattractant for the highly metastatic murine Lewis Lung carcinoma cells (please see

Chapter VII). - Tumor invasion and metastasis are not simply the outcome of mechanid pressure

exerted by the enlarging tumor mas, but rather the cumulative effect of multiple changes

in gene expression. Invasion d t s h m an imbalance and deregdation of positive and

negative signals which are tightly controlled under normal physiological conditions (7).

During nomial physiological pmcesses such as migration of smooth muscle cell fkom the

media to the intima (262), angiogenesis (230), embryogenesis and morphogenesis (263).

nerve growth cone extension and homing (264)- and tmphoblast implantation (265).

Li Long. Regdarion of Tumor Ceff 1-ion and Mktastasir by the Typr I Iiuulin-fi& Growth Factor Renptor (ZGF-IR)

Cellular motility and invasion are tightly regulated and the processes terminates when the

stimulus is removed. Thus for example, angiogenesis will cease when the source of the

angiogenic factor is removed or the concentration of angiogenic factors diminished (230).

Neurite migration and invasion are arrested when microenMronmentai signals "alert" the

neurites to destination arrivd (264). Diffetentiation of trophoblasts coincides with

termination of invasion (265). in contras& invading tumor celis appear to have lost the

control mechanisms which prevent normal cells fiam invading neighboring tissue at an

inappropriate time and space (7).

If one assumes that the malignant tumor ceii is inappropnately expressing cellular

mediatoa of invasion n o d y "silenced" in the non-malignant precursor ceiis, thni the

fuodamentai difference bctweni normal and maligaant ceii must be at the level of

regdation of these mediators. These implies that regulatory proteins which can activate

or downregulate the invasion are inappmpriately e x p d (266). A major goal,

therefore, is to understand which signais and signal transduction pathways are

maintaining mediators of ceii migration and invasion in a constitutively active fonn.

The Insulin-like Growth Factor I Receptor: Structure, Function and Role

In Malignancy

Li Long. Regu&tion of Timor Ccll fmtcuion anà ,cAc1~1141is by I/Y / J ~ ~ l ï n - i i k e Growth Factor Receptor /fGF-IR)

2.1. The type 1 insulin-lüre growth factor meptor (IGF-IR)

IGF-ZR was originally isolated fiom human placenta (267). Structuraily it was found to

have a strong similarity to the iasulin receptor but it binds iasulin with a relatively low

affinity (268). The human IGF-IR cDNA was cloned in 1986 by Axe1 Ullrich and

colleagues (269). It consists of an open reading fiame of 4101 nucleotides that encode a

protein of 1367 amino acids. The rat IGF-IR cDNA bas recently been cloned and

sequenced (270).

The IGF-IR is synthesizeâ as a single polypeptide chah which is then glycosylated and

proteolytically cleaved at a tetrabasic arg-lys-arg-arg sequence at position 707-710 into

a- and B- subunits (269). The receptor wmists of 2 a and 2 f3 chahs, with severai

alpha-alpha and alpha-beta disuifide bridges. The ligand binding domah is located on the

extracellular -alpha- subunit. Approximateiy a 1/3 of the (3 subunit is extracellular and is

comected to the intraceIlular portion by a single trammembrane domain. The tyrosine

kinase catalytic site and the ATP-binding site are locaîed on the cytoplasmic portion of

the B subunit (59).

The IGF-IR is expressed in nearly al l tissues and on cells in culture (59). Northem

analysis of IGF-IR mRNA reveals a major transcript of 11 kb with a minor transcript of

7 kb in some tissues (269). The liver has exceedingly low levels of IGF-IR mRNA.

However, receptor mRNA expression aud IGF-I binding sites increase during liver

regeneration and can also be found in fcîaî rat bers (271,272).

The molecular weight of the a and B subunits of the insulin receptor have been repted

to be between 130,000 and 135,000 and between 90,000 and 95,000, respectively (273)

(274). Similar molecular weights have been rcported for the IGF-1 receptor subunits (275)

(267), although there are dinérences between the molecular weights of the two

receptoa. For example, the subunit of the IGF-1 receptor h m IM-9 celis nuis on SDS

gels as a broader band with a siightiy higher molecular weight than that of the insuLin

receptor h m the same cells (276). Recent cloning of the iasuün receptor (36) and IGF-1

receptor (269) cDNA has, however, made it clear that although they share about 60%

homology at the amino acid level, they clearly are derived h m different genes, which,

perhaps surprisingiy, are located on different chromosomes (chromosome 19 for the

i d i n receptor and chromosome 15 for the IGF-1 receptor). The physiological properties

of the IGF-1R and insuLin receptors ciiffer substanttidy. 'Activation of the IGF-1R is

primarily mitogenic whereas the insulin receptor induces principally metabolic eEects

(59)-

2.2. The IGF-1R Iigands

The IGF-IR Ligands include IGF-1, IGF-II and insulin. The receptor binds IGF-1 with

high afWty (Kd 1nM) and IGF-II and insulin with considerably lower affinities (10 -fold

and 100-fold lower affTnities, respectively) (269). The gene and protein structures of IGF-

1 and IGF-II have been extensively studied. IGF-1 and IGF-II share about 70% sequence

homology and have close structural homology to insuiin. The mature 70 amiw acid IGF-

1 peptide, Like insulin, has an A and B domain. In addition, a C domain connects the A

and B domains, and a unique 8-amino acids D domain is present at the carboxy-terminal

end (277,278). The human IGF-I gene is located on chromosome 12 and is composed of

5 exons that are differently spliced to form two mRNA molecules, IGF-Ia and IGF-Ib

(279). Mature IGF-II also contains 4 domains (BCAD) and is 67 amho acids in Length.

IGF-1 plays a important role in the regdation of the dl cycle, in cellular transformation

and in maintenana of the dgnant phenotype (1 1). The induction of DNA synthesis

and mitosis generally require more than one growth factor. The effect of growth factors

on the ceU cycle has been most closely studied in the rnouse fibroblast line Balb/c 3T3

(280). In these celis, the induction of cornpetence, i.e., the ability to enter S phase, can be

mediated by several p w t h factors including platelet-derived growth factor (PDGF),

fibroblast p w t h factor (FGF), and epidermal growth &or (EGF). However,

progression through G1 into S phase will not occur without IGF-1. An intact IGF-IR

appears to be requind for at l e s t some of the biological effkcts of both the EGF and

PDGF receptors (281, 282). Thus when the human EGF receptor was overexpressed in

IGF-IR-deficient mouse fibroblasts, EGF treatment did not stimulate DNA synthesis,

cellular proliferation, or colony formation in sofi agar above background levels (28 1).

These defects were comted by expression in these cells of the IGF-IR, and EGF then

became effective (281). Similarly, overexpression of the PDGF b-receptor in cells lacking

the IGF-IR did not lead to ligand-activated proliferation or growth in sofi agar (282).

Again, expression of IGF-IR corrected these deficiencies (282).

2.3. The IGF Binding Proteins.

The physiological activities of IGF-1 are modulated by their association with the IGF

binding proteins (IGFBPs). To date, six IGFBPs have been identifïed comprising a

structurally related family of ~ecteted proteins that bind both IGF-1 and IGF-II with high

affinity (283-286). The IGFBPs Vary in length h m 216 to 289 amino acids and are

composed of s h d cysteine-rich amino- and carboxy1-terminal domains (286). In

contrast, the central portion of each IGFBP is unique (286). The IGFBP genes have a

simple and conserveci structure, and each gene is transcribed and processeci into one or at

most a few mRNAs (286).

The IGFBPs are modulators of IGF action. The IGFs present'in the blood and other

bioiogical fluids are bound to IGFBPs, and this interaction maintains reservoirs of these

growth factors in the circulation and elsewhere (284, 287). In conjunction with another

protein, termed the acid labile subunit (ALS), IGFBP-3 is primarily responsible for

maintainhg IGF levels in the blood (287,288). Other IGFBPs found in the bloodsfream,

including IGFBP-1, -2 and -4, can cross endothelid barriers and thus may transport IGFs

fiom the circulation to peripheral tissues (284, 286, 287). Severai IGFBPs are found in

the extracellular environments of many tissues and may regulate IGF accessibility to

receptors andor provide a I d storage depot The local hmctions of IGFBPs may be

modulated by interactions with the extracellular matrix and with the ceIl surface. In

addition, local levels of several IGFBPs are modified through specifk proteolysis (52,

Li Long. Regdation of T i o r Ceil inwuion a d .Uerusrarir by r k Typc I Innrlin-like Growrh Factor Rccepror (IGF-f R)

a 289). Extra-ceiidar matrix (ECM) -associated BP such as IGFBP- 5 have been described

(290). The association of BP-5 with the ECM, provides a mechanism for iGF-1 binding

and sequestration in the ECM where it can be exposed in processes such as wound

heding, tissue repair and tumor celi invasion.

The roles of individual IGFBPs in growth and development have not been examined in

detail, although a wealth of studies exists in tissue culture systems, as summarized

recently (284, 287). Transgenic mice overcxpressing IGFBP-1 showed mild growth

retardation and modest hypergiycermia (291), mice lacking IGFBP-2 by targeted gene

disruption were reported to be normal (292) and overexpresion of IGFBP-3 caused

selective increases in heart, liver, and spleen weights (293).

One fimction of IGFBPs is to buffer the effective concentrations of IGFsy which are

present at combined Ievels of about 750 ag/ml in serum, concentrations which are far

beyond their mitogenic thresholds. The h&life of semm IGF-1 depends upon its

association with the IGFBPs. The presentation of IGF-1 to ceiis by IGFBPs may account

for reported hdings that IGFBPs altematively enhruice or inhibit the mitogenic potency

of IGF-1 (294). For example, exogenous addition of IGFBP-1 blocked the mitogenic

effect of IGF-1 on human breast cancer MCF-7 ceus in one report (295) while another

report descnbed the reverse action for IGFBP-1 (296). The fÙnction(s) of the BPs are

not as yet fully understood. The experimental evidence suggests that they c m act to

Li Long. Regulo~mt of Tumor Cell l'ion and Metamsis ëy h e Typc I Inmfin-likr Gm wih Factor Receptor IIGF-IR)

a stabilize senun concentrations of IGF- 1, potentiate IGF- 1 function by facilitating the

ligand/receptor interaction or inhibit it by forming inactive complexes (297).

These actions can be summarized as foiiows:

1. transport IGFs h m the circulation to peripheral tissues

2. maintain a reservou of IGFs in the circulation

3. potentiate or inhibit IGF action

4. mediate IGF-independent biological effects

2.4. IGF-IR- Signaüng:

While signaling via the insuiin receptor has been a subject of active investigation for

several years, structure-fiundon analyses of the IGF-IR have ody rrcently begun to shed

light on the signaling pathway activateci upon ligand binding by this receptor (298).

Current analysis of the events thst follow gmwth f w r receptor activation comprises

both the identification of the cytosolic and nuclear mediators that lead to transcriptional

activation and the subsequent expression of mgdatory gene products. The evidence

currently avaiiable indicates that upon Ligand bînding the f3 subunit of the receptor is

autophosphorylated. This leads to phosphorylation of a cytoplasmic protein of - 165- 195

kDa (molecular mass varies in different reports) which was collectively termed the pp185

IRS-1 (innùin nccptor substrate-1)(299-301). This protein although it does not contain

SH2 or SH3 domains, has potentiai biading sites for SH2- containùlig proteins and

appears to act as a multi-site "docking" protein serving as a link between downstream

Li Long. Reguhtion of Tumor CeIf I . i m anâ ,Wtm&zsis by I/r Typr I Id in - f i& Growili Factor Receptor (fGF-IR)

substrates and the receptor. One of these substrates is the signaiing pathway intermediate

protein kinase C (PKC) (302). Recently the nuclear proto-oncogenes c-Jun and c-Fos

which had been identified as reguiaters of collagenase (MMP-1, MMP-3, MMP-9, and

MMP-10) were reported as nuclear substrates downstream of the IGF-IR signaiing

pathway suggesting that some of the receptor bctions are mediated via transcriptionai

activation of AP-1 binding sequences (303, 304). IRS- 1 dso associates with other SH2

dornain-containing proteins involved in growth f ~ t o r sipnaling pathways, inciuding Grb-

2, Nck (also an adaptor protein), and Syp (also known as PTPlD, PTPX, or SHPTP2, a

phosphotyrosine phosphatase) (305). Syp b m e tyrosyl-and threonine-phosphorylated

in response to PDOF and EGF stimulation (306). Syp is also lmown to associate with

IRS-i via its SEI2 domain which interact with specific phosphotyrosine-containing

sequences of IRS-l(307).

Another downstrearn substrate of insuiin and IGF-I receptor signaîing is the Shc (src-

homology/collagen) protein (308). The Shc family is comprised of three proteins of 46,

52, and 66 kDa thai contain a single SH2 domain (309). Like IRS-1, upon tyrosine

phosphorylation, Shc associates with Grb2, and subsequently activates Ras via a Grb2-

mSOS cornplex.. Since the Shc gene has transfomiing pmperties (309), it may play an

important role in the transfomiing ability of the IGF-1 receptor. The IGF-IR also directly

phosphorylates Crk (3 IO), a cellular homologue of v-crk. Members of the Crk family bear

SH.2 and SH3 domains, share homology with Grb2 and Nck, and interact with the ras-

binding protein mSOS (311). The relative importance of this pathway in signal

transduction by IGF-IR rernains to be elucidated.

Analysis of deletion and substitution mutations within the p-chain of the receptor has

yielded additional new information about IGF-IR function. As expected, the data

demonstrate that receptor tyrosine kinase activity is required for rnost signaihg bctions.

A mutation of lysine-1003, the ATP binding site, wmpletely disrupts receptor h c t i o n in

transfected NIH 3T3 fibmblasts (312). Minimal tyrosine phosphorylation occurred in

response to IGF-1, and Ligand binding did not trigger activation of PU-Linase, glucose

uptake, or DNA synthesis (312). Receptors containhg Dchain tnincated at amino acid

952, and thus Iaclring the kinase domain, did not transmit growth-promoting signals and

acted as dominant-negative inhibitors when transfected into ceUs expressing wild-type

receptors (313)A sidarly nonfimctionai receptor was created by substitution of

tyrosines- 1 13 1, -1 135, and - 1 136 with phenylalanine (3 14, 3 1 5). Alterations of a i l three

tyrosines, which are the major sites of receptor autophosphorylation in the IGF-IR,

resulted in no autophosphorylation, no phosphorylation of cellular substrates, including

iRS-1 and Shc, diminished ligand-activated receptor internalization, and no short-,

medium-, or long-term biological effêcts (314, 3 15). Mutation of individual residues in

this triple tyrosine cluster also caused a decrease in the extent of autophosphorylation and

diminished IRS- 1 and Shc phosphorylation (3 16-3 1 8). While substitution of

phenylalanine for tyrosine at residues 113 1 or 1135 did not inhibit induction of DNA

synthesis or cellular proliferaton in q n s e to IGF-1 in receptodeficient fibroblasts

MAP Kinase Kinases (MEKs)

MAP Kinases S6 kinase

(ERKs) \ /

Other Substrates -, l,#-,.,

YPKC signaling

- 0 . - e - Transcription Factors

Fig 2-1. Schemaîic representation of intncellular sigaaluig pathways of the IGF-1 receptor. Upon binding IGF-1, the IGF-1 receptor undergoes autophosphorylation at multiple tyrosine residues. The intrinsic kinase activity of the receptor also phosptiorylate IRS-1 at multiple tyrosine residues. Various SH-domaincontainhg proteins, including PI3-kinase, Grb2, Syp, and Nck, associate with specific phosphotyrosinetontaining motifs within IRS-1, as shown. Activation of IGF-1 receptors also results in tyrosine phosphorylation of Shc, which then cornpiexes with Grb2. Grb2 is tightly associated with the "an guanine nucleotide exchange factor SOS, which activates Ras. IGF-I c m apparently activate Ras via b t h the ES-l-Grb2-SOS or the Shc-Grb2-SOS pathways. This leads to the activation of a cascade of protein kinases including Raf-1 and one or more reiated kinases, MAP kinase kinases (or MEKs), the MAP kinases, and S6 kinase. These protein kinases, in tum, activate various other elements. including nuclear transcription factors. Alterations in expression of various IGF-I-responsive gens results in longer term effects of IGF-1, including growth and differentiation.

Li Long. Regdarion of Timor Ceff f l l ~a~ ion curd Meranasis by the Typc I I d i n - i i A r Growth Facror Recepror 1EF- IR )

a m f e c t e d with each mutant, modification of tyrosine-1 136 did reduce replication (3 17).

In contract, each substitution mutation blocked colony formation in soft aga. Taken

together, these results dernonstrate that each tyrosine in this cluster is not equivalent and

indicate that a M y tyrosine phosphorylated and presumably hilly functional receptor is

required for anchorage-independent growth but not for mitogenesis.

Tyrosine-950 is essentiat for binding and phosphorylation of IRS- 1 (3 19-32 1).

Substitution of phenylalanine for tyrosine at this site did not alter autophosphorylation or

ligand-activated teceptor intemakalion, but blunted phosphorylation of IRS-1 and

blocked other biological effects (320, 321). This tyrosine also has been found to bind to

another signahg intemediate, the rar GTPase-activating protein (GAP) (322). More

extensive mutations, deletkg residues 947-950 or 944-965 of the juxtamembrane domain,

additionally abrogated IGF-1-stimulated receptor internalization (3 19,32 1).

Mutation of tyrosine-1 3 16 near the carboxyl-terminal of the IGF-IR also interrupts

protein-protein intefactions between the activateci IGF-IR, the regdatory subunit of PI3-

kinase p85, and the tyrosine phosphatase SH-PTP2 (322, 323). The fbctional

consequences of these mutations have not been elucidated.

Two tyrosines at positions 1250 and 1251 of the IGF-IR are not found in the insulin

receptor (269). Substitution of either or both amino acids with phenylalanine had a

minimal effêct on autophosphorylation and substraîe phosphorylation in receptor-

Li Long. Regdation of Tùmor Celf Invasion and Merastaris by the T j f I d i n - l i k e Growrh Factor Rr~cpror (IGF-IR)

a deficient fibroblasts traasfwted with these mutants, and did not alter cellular proliferation

in response to IGF-1 (324). In contrast, the double mutation or substitution for tyrosine-

1251 alone caused a profound decrease in the efficiency of cellular tradormation, as

measured by diminished colony growth in soft agar (323,324). these results demonstrate

that the mitogenic and transforming properties of the IGF-IR cm be dissociated and

present an experimental mode1 to test the hypothesis that distinct signal-transduction

pathways mediate each biologicai effcct,

Additional determinants within the carboxyl-terminal tail of the IGF-IR may be involved

in mediating cellular bransformation. Mer trardection inîo receptordeficient fibmblasts,

receptoa lacking the last 108 amiw acids showed ligand-dependent autophosphorylation,

mediated phosphorylation of IRS-1, activated PI3-khase, and stimuiated cellular

proliferation, but did w t enhance colony formation in sofi agar (325). These studies also

indicate that signal transduction pathways responsible for the mitogenic and CransfoCIlling

properties of the IGF-IR may be separable.

2.5. Role of IGF-1 in Cellular Proliftration

The G1 phase of the ceU cycle is the interval when ceUs prepare for DNA synthesis (S

phase), which is marked by chromosoma1 replication. Studies with mouse fibroblasts

have demonsirateci roles for several growth factors in progression of the cell cycle nom

G1 to S (14). A puise of PDGF is required for fibroblasts to becorne competent to move

into G1, and either EGF or IGF-1 were found to be necessary for subsequent progression

Li Long. ReguIatron of Turnor Celf Invasion and r K e ~ ~ m L t by the rbpct I Illsulin-ii& Growrh Factor Reccpror (IGF-IR)

0 into S phase (14, 280). Pardee (14) also identifïed a point in Gl termed V, which was

opentionally defined as the halting stage observed in the absence of essential amino

acids. In addition to amino acids, which are necessary for protein synthesis, only IGF-1

was s h o w to be required for cells to move past V into S phase (14,280).

The range of ce11 types that require IGF-1 for survivl and growth is broad. It includes

fibroblasts, hematopoietic cells, smooth-muscle ceils, keratinocytes, osteoblasts and

chondrocytes, ~manrmary epithelid cells, thyroid ce&, and neutoglial celis (326). More

criticaily, recent obmations have demonstraied that a fundional IGF-IR is needed for

many aspects of normal cell cycle progression (327). In fibroblasts derived h m mouse

embryos with a nuU mutation in the IGF-IR, aii phases of the ceil cycle were slowed, and

0 doubling time after incubation in medium with 1W serum was proionged h m 44 h in

wild-type fibroblasts to 109 h in celis h m receptor-deficient mice (327). In addition, cell

cycle progression could not be stimulated to a normal rate in these fibroblasts by

expression of dominant oncogenes (327, 328). These vitro results potentidy provide

an explmation for the marked growth rebrdation seen in mouse embryos lacking the

IGF-IR (329), since delayed cell replication and consequently diminished ce11 number

codd be responsible for the dwarfism that results h m this mutation. These growth-

promoting effects of IGF-1 contribute to its role in tissue repair and regeneration (330).

IGF-I is also a survival factor rescuing responsive cells nom apoptosis. This effect is

thought to be independent of its mitogenic potential(33 1). In addition, IGF-1 was s h o w

to support the viability of non-proüferating cells, notably neaons, in culture (332).

Li Long. Rcgrcfut~on of Twnor Cell fhvation a d Lkt011cuu by the Typc I fiuufin-lïkc Growth Factor Recepror (IGF-IR)

2.6. Role of IGF-I and IGF-IR in ceii death

IGF-1 has been shown to hct ion as a Survivai fa ton for various ceIl types including

myoblasts, neurons, and oligodendrocytes. Both IGF-1 and PDGF have k e n found to

blunt apoptosis induced by c-Myc in seru~~l-deprived fibmblasts, and this effect was

independent of celi cycle progression or ongoing protein synthesis (333). In other studies,

IGF-1 and the IGF-IR were show to be requved for Survivl of cultured hematopoietic

ceils after trophic fiictor withdrawal (334). IGF-1 could dso prevent apoptosis in

fibroblasts exposed to the topoisornerase inhibitor etoposide (333, 3 3 5) and block the

death of a variety of turnor celis cultured for a short tenn in vivo (33 1,336).

The molecdar b i s of the anti-apoptotic effect of IGF-1 is poorly uaderstood (59,

3 35). 1GF-i could prevent apoptosis induced by overexpression of the c-myc oncogene in

rat4 fibroblasts (333). It could also inhibit apoptosis of IL-34ependent hematopoietic

cells following removal of IL-3 (334). The anti-apoptotic role of the IGF-IR in vivo bas

recently been examinecl (336). Several tumor lines expressing antisense IGF-IR RNA are

non-tumorigenic possibly because they die rapidly upon injection into animals (33 1, 335,

336).

2.7. IGF-IR and maügnancy.

Overexpression of the IGF-IR in fibroblasts tedted in the acquisition of tumorigenicity

when the transfected cells were injecteci into immunodeficient nude mice (337). This

0 transforming effect could be blocked by expression of a tnincated receptor lacking a

tyrosine kinase domain (313, 316, 317), possibly due to formation of heteromerk

complexes consisting of both wild-type and mutant ap-heterodimers. Fibroblasts lacking

the IGF-IR also do not form tumors in nude mice (327), nor do cells expressing receptors

with mutations in the tyrosine cluster at amino acids 1 13 1, 1 135, and 1 136 (3 14-3 16), or

the carboxyl-terminal tyrosine4251 (323, 324), thus indicating that intact receptors are

required to mediate tumorigenesis.

C6 rat glioblastoma ceUs form rapidly progressing malignant neoplasms when injected

into irnmunodeficient mice or syngeneic rats (331, 338). Cells pretreaîed with IGF-IR

antisense oligonucleotides fonned smaller tumors than controis (33 1). A decline in tumor

a ce11 survival in vivo aiso was observed in Cd giioblastomas stably expressing an IGF-IR

antisense cDNA (336).

The IGFs are potent mitogens for a wide range of hunor celi types in vitro. Activation of

IGF-IR can promote turnor celi growth by autocrine or paracrine mechanisms (339).

IGF-1 or IGF-IR expression have k e n documented in many hunor types including lung,

breast, thyroid, gastrointestinal tract, liver, pancreas, kidney and neuroendocrine tumors

(339). Several oncogenes have now been shown to affect IGF-1 and IGF-IR expression.

Oncogene c-niyb induces the expression of both IGF-1 and IGF-IR, and c-myc may also

increase the expression of IGF-IR (340). Overexpression of IGF-1R has in tum been

shown to induce cellular traiisformation f i vitra and pmmote colony foxmation in sofk

agar (337, 341), the &I v i t r ~ correlate of the tumorigenic phenotype. Reduction of IGF-

1R expression has been shown to inhibit cellular transformation and tumorigenicity as

mentioned above (342-344). Anti- IGF-1 (R) reagants suc6 as IGF-1 analog, antibodies

and antisense strategy have shown the potentid therapeutic application in the fûture (328,

Li Long. Regulorion of Timor Ccll l'-ion and Metosfaru by the Typr I Insuiin-il& Growih FPEIOI R r ~ p t o r (IGF-I R)

Chapter III

Matrix Metalloproteinases in

Cancer Invasion and Metastasis

Li Long. Rcguhion of Timor C d l'ion and ~Ue&asmis by the Type I Imiin-iike Growth Factor Receptor (IGF-IR)

Turner invasion and metastasis entail multiple specific interactions between the tumor

cells and the extracellular ma& (ECM, see r d 7). Ody metastatically comptent cells

can successfiilly navigate through these interactions. Initially, tumor ceU escape h m the

primary tumor is thought to require decreased adhesiveness to 0 t h tumor cells or ECM

proteins. However, the arrest of neoplastic cells in a target organ which leads to organ-

colonization is thought to be mediated by specific tumor-endothelid cell interactions and

adhesion to subendotheiid ECM proteins (145, 348). Attachment to ECM proteins may

be the trigger for ECM degrdation - a key step in -or invasion and metastasis.

The proteolytic enzymes or hydrolases which have b a n implicated in tumor invasion and

metastasis can be classified into at least five groups on the basis of their active site,

optimal pH, cation dependency and as a consequeme of the above, susceptibility to

specific inhibitors (349). These include the sezinc proteinases, cystein proteinases, the

aspartic proteinase cathepsin D, metallopmteinases and glycosidases. Of these groups, the

most extensively studied have been the serine proteinase uPA and plasmin and the matrù<

metalloproteinases (122).

The matrix metalloproteinases arc a family of ME - dependent hydcolases with well-

characterized structurai and catalytic properties (350). They share varying levels of

homology, and cm be inhibited by metd chelators or a family of endogenous inhibitors

known collectively as tissue inhibitors of metailoproteinases (TIMPs)(351). The

metalloproteinases are secreted as proenzymes that undergo proteolytic cleavage at an

e amino-ter- domain during activation. There are eight well-characterized members of

the family : interstitial collagenase, neutrophil collagenase, stromelysin- 1, stromelysin-2,

stromel ysin-3, matrilysin, gelatinase A and gelatinase B (1 22, 3 52). Additional members

have been identifid recently including collagenase 3 (353), metalloelastase (354) and

membrane-type mattix metalloproteinase (MT-MMP) (355).

The collagenases are zincdependent endopeptidases with a broad spectnim of proteolytic

activity against scveral components of the ECM (123). They include intersritid (type 1)

collagenase (MMP-1), coiiagenase-3 and type IV mllagenases (gelatiuases).

MMP-1: also known as Type 1 coiiagenase initiates the breakdown of type 1, II and III

0 fibnllar coilagens which together constitute the most abundant proteins in the body (356).

It therefore plays a critical role in tissue remodeling which occurs uuder both

physiological aud pathological conditions. Clesvage of all three coiiagen alpha c h a h

takes place at a single locus by hydrolysis of the peptide bond between residues 771 and

772 which is located immediately following a Gly residue. In addition, MMP-1 can also

promote the activation of MMP2 (357). The synthesis of this collagenase can be induced

by a variety of mediators including cytokines such as IL-1 and TNFa, growth factors

such as EGF, PDGF, and bFGF and chernical modulators such as phorbol esters,

cytochalasin B. and calcium phosphate crystais (96,358,359).

Li Long. RPgula~ion of Twor Cell Invusion ami Metastarir by the T m IInrrrlin-Me G r o h Factor Receptor flGF-I R)

MMP-2: MMP-2 is Also known as the 72 kIh type IV collagenase and gelatinase A

The major substrate for MMP-2 is type IV collagen - the major protein of basement

membranes. It has also been implicaîed in the degradation of type V, VI1 and X

collagens, gelatins, elastins and fibronectin (356, 360). Activation of the 72 kDa

proenyme by agents such as organommurials resuits in the autoçatalytic cleavage of an

amino-terminal domain giving rise to two activated forms of 66 and 62 kDa each (361).

The factors which regdate synthesis of this emyme have not been M y elucidated. TGFP

has k e n shown to induce expression of this eayme (81). However, it appears to be

rehctory to stimulation with photbol esters, IL-1 and EGF - factors which stimulate

synthesis of MMP-1. A recent study has shown that expression and secretion of this

enzyme could be positively regulated by ligation of the vitronecth receptor (362).

MMP-9: (also known as the 92 LDa type IV coüagenae and gehtiaase B) is similar to

MMP-2 in both structure and substrate specifkity (356) but is diaerently regulated (363).

h o n g the known substrates for this e q m e are gelatin, elastin, fibronectin,

proteoglycans, laminin and types IV, V, VII and X collagens (356,363). Transformeci or

maiïgnant celis and rheumatoid synovial fibroblasts Wuently express the enyme (364-

366). Ln these cells the expression of MMP-9 can be stimulated by inducers such as

phorbol esters, IL-IB, EGF and TNFa (363,367).

The activation and hct ion of the collagenases are tightiy regulated by a family of

m inhibitors known as the tissue inhibitors of metaHoproteinases (TIMPs). Of particular

Li Long. Regufation of T'or Ce11 i ï i i o u and Merastasu by the Typc I Iiuuiin-iikc Growtk Factor Rcceptor (IGF- I RI

relevance to the collagenases are TU.IP-1 and TIMP-2. While TIMP-I is generic and cm

inhibit the activities of several MMPs incIuding the collagenases and stromlysins (368).

TIMP-2 is more specific and inhibits mainly MMP-2 and MMP-1 (369).

The ro1e of metalloproteinua in tumor invasion and metastasis

The role of collagenases in tumor invasion and metastasis was orighally postulated on

the basis of observations, made as early as 1980, that metastatic tumor cells have

increased levels of type N collagenase as compared to the non-metast;itic counterparts

(370). This was nrst described by Liotta in the context of non-metastatic and metastatic

variants of BI6 melanoma ceils (370). The role of MMPs in tumor progression and

metastasis was also supporteci by the fïnding thai rat embryo fibroblasts (REF) which

were transformeci with both the rar and myc oncogenes were metastatic and expressed

high levels of ''type IV collagenase activity" (371). REF cells transformed with ras and

Ela, on the other han& were found to be equally turnorigenic but non-metastatic and

expressed low levels of ''type N coilagagenase activity". Subsequent studies identined this

"activity" as mediated by MMP-9 (372) andfor stromelysin-1 and -2 (373) . Studies

comparing the activity or production of MMPs in ceil Lines isolated h m lesions of

different stages of malignant disease have led to the conclusion that enhanced or de novo

MMP production is assaiiated with advanced stages of many cancers. For example,

MacDougall et al have found that cell lines denved nom advanced melanomas, that is,

ce11 Iines derived from lesions in which the patients succumbed to their disease. were

vimially 100% positive for expression of MMP-9 (374). On the other hand ceil Lines

a derived fiom early melanomas, i.e. those lesions that redted in a cure subseqwnt to

removal, were uniformly negative for MMP-9 expression Colon carcinama ce11 lines also

tend to produce increased amounts of MMP-2, although many tumor cells seem to be

uniformly positive for MMP-2 regardless of tumor stage or grade (375). hterestingly, it

has k e n found that prognosis in breast cancer correIated not with the expression of

MMP-2 but with its level of activation and that the presence of activateci MMP-2 was an

indicator of more aggressive lesions (376). Additional studies in this system revealed

that the activation of MMP-2 wuld be l o d i z d to the membrane k t i o n of breast

cancer cells (376). in addition, a marked reduction of TIMP production by malignant as

compared to benign cells was reportml in murine fibmblastic tumors (128). Plasma

membranes isolated h m human and mouse cancer cell lines were found to be highly

enriched in gelatinolytic and wllagenolytic açtivities (377). Increased collagenolytic

activity was also o b s e ~ e d in various mesenchymal and epithelid tumors (378).

Stimulation of the %h integrin receptor enhanced MMP-2 pduction and stimulated

melanoma ceU invasion (8 1) .

Much of the deikitive evidence for a d e of MMPs in the progression and metastasis of

tumors cornes fiom studies aimed at modulating the levels of MMP inhibitors. Work

fkom several groups has shown that transfection of cancer celis with the genes for either

TIMP- 1 or TIMP-2 had marked effects on their invasiveness in vitra or metastatic spread

in vivo in experirnental animals (379). Similar d t s were obtained following

administration of TIMP-1 or synthetic MMP inhibitors (380).

A growing number of reports, however, suggest that MMPs may also &ect the growth of

tumor cells at the primary site. This effect was initiaily described by Khokha et al for

Swiss 3T3 fibroblasts which were rendered tumorigenic as well as metastatic when

transfected with a TIMP-1 antisense expression vector (381). Using intravïtai

videomicroscopy, Koop et al. have found that overexpression of TIMP-1 in BI6

meianoma cells did not &èct the ability of ceus to extravasate but did teduce tumor celi

growth foilowing extravasation (382). Similady, DeClerck and ~oiieagues found that

transfection of melanoma cell with TIMP-2 cDNA reduced theu p w t h and metastasis

(383). On the other hand transfection of colon carcinoma celis with matrilysin cDNA

resuited in i n d tumongenicity (384) and the treatment of colon carcinoma or

ovarÏan carcinoma -karing mice with the synthetic MMPs inhibitor BB-94 r ed ted in a

ciramatic reduction of both tumor burden and metastasis (385). Taken together, these

studies strongly suggest that MMPs play a role not only in invasion but also in growth

regdation.

WhiIe the role of MMPs in facilitating invasion can be attributed to their ECM-

degrading function, the mechanisrn underlying their d e in p w t h enhancement at the

primary site is not clear. MMPs may facilitate the expansion of a growing tumor mass by

mediating degradation of the stroma1 ECM. They may activate latent growth factors or

inactivate growth inhibitory molecules indirectly Muencing the growth of tumor cells

(386). They may dso be involved in angiogenesis and thereby promote tumor

progression (386). In al1 of these processes, the MMPs may be produced by the tumor

cells or by adjacent stroma1 cells responding to tumordenved factors and contributhg to

the increased proteolytic activity in the tumor microenvironment.

Chapter IV

The Role of lnsulin-like Growth Factor I in Metastasis:

Studies with the Lewis Lung Carcinoma Model

Li Long. Reguhrion of Tumur Celf Invos~on and .Wewtaris by I/w TF I Idin-Iikc Growrh Factor âeceptor ffGF- IR)

0 4.1

Results of my studies on the role of the insulin-iike growth factor receptor system in

cancer metastasis are presented in part in this chapter in the form of two published papers.

I was responsible for al1 of the experimental work described in this chapter. The plasmid

vector expressing antisense IGF-IR cDNA was a gift h m Dr. Renato Baserga and

colieagws at the Jeffmon Cancer Institute, Thomas Jeffetson University in Philadelphia.

Al1 of the studies were carrieci out with the murine Lewis lung carcinoma model which

consists of two subiines with divergent metastatic pmperties namely, carcinoma H-59

cells which are highly and preferentiaiiy metastatic to the liver and M-27 ceils which are

moderatel y and specifidy metastatic and to the lung (Please see table 4- 1).

in the nrst paper Cong, L., Np, J., and Brodt, P. Paracrine growth stimulation by

hepatocyte-derived insulin-lüce growth factor-1: A regdatory mechanism for carcinoma

cells metastatic to the liver. Cancer Res. 54: 3732-3737, 1994), H-59 and M-27 cells were

used to study the mie of paracrine factors in regdating the ability of these tumor ceiis to

form liver metastases. H-59 celis express significantly higher levels (5-fold) of IGF-1

receptor than M-27 cells (Fig Sc in paper). Hepatocyte conditioned medium (HCM) was

found to be mitogenic for H-59 but not M-27 cells whereas lung tissue conditioned

medium (lung CM) was mitogenic for both celi lines (Fig 1 in the nrst paper), raising the

possibility that target organderived growth fmoi(s) can regulate organ specific

a dissemination in this model. Subsequently it was found that IGF-1 was mitogenic for H-

a 59 but not M-27 cells whereas other growth factors tested such as EGF, PDGF, HGF,

and TGFa did not have differential effects on the two ce11 lines. Furthemore, the

rnitogenic effécts of HCM on H-59 cells could be depleted by a monoclonal antibody to

IGF-1, ident-g IGF-1 as the HCM growth factor. Western blot analysis connmied the

presence of IGF-1 in HCM.

In the second paper (Long, L., Rubin, R, Baserga, R, and Brodt, P. Loss of the

metastatic phenotype in murine carcinoma cek expressing an an- RNA to the

Win- l ike growth factor receptor. Cancer Res. 55: 10061009, 1995), the d e of the

IGF-I receptor in metastasis in this mode1 was M e r hvestigated using H-59 ceiis

tramfected with a plasmid vector expressing IGF-IR cDNA in the antisense orientation.

The antisense transfectants lost their responses to IGF-1 and HCM in vitro and their

metastatic potentiai in vivo.

9 eview O- *

Sublines H-59 and M-27 were originally derived h m metastatic colonies to lung (M-

27) and Liver (H-59) which developed in animais bearing a ss. Lewis lung carcinoma

(3 LL) tumors (3 87). The spontaneous hepatic and pulmonary metastases were removed

and serially implanted S.C. (seven or more consecutive implantations) in the axillary

region of new recipient mice until a stable pattern of organ-selective @ver and lung,

respectively) metastasis was established.

Li Long. ReguIation of Timor Cell Imasion and M e t a s ~ i s by üte T ~ p c I Idin-Iïke Growrh Factor Recepror (IGF- I R)

Common and unique properties of sublines H-59 and M-27 cells:

Summary of previously pubüsheà data

Both tumor sublines rrtained the characteristic morphology of the parent line originaliy

described as a "poorly differentiated epidermoid carcinoma" (3 87, 3 88). No significant

differences were found between the incidence or rate of growth of these two tumor celis

in vivo foliowing S.C. inoculation. However M-27 ceUs are moderately metastatic

specifically to the lung, while H-59 ceiis are highly metastatic predominantly to lymph

nodes and liver but also to lung, adrenal gland, kidneys and even the heart. In this model,

tumor cell adhesion to cryostat sections or cultwed cells denved h m the preferred target

organ of metastasis in vitro was found to correlate with their patterns of organ selective

a metastasis in vivo. Thus tumor H-59 cell adhered significantly better to cryostat sections

of lymph nodes and isolated hepatocyte monolayers than M-27 celis suggesting that celi

adhesion may play an important role in organ-selective meuistasis in this model (388,

389). Using a monoclonal antibody specifk to H-59 cells @Wb C-11), a Mr 64,000

plasma membrane glywptein was identifieci on H-59 cells and which is involved in the

adhesion of the tumor celis to hepatocytes (390). ~rea&ent of H-59 celis with this

monoclonal antibody markediy and specincally reduced the ability of the cells to fomi

hepatic metastases (391). In addition, Merences were noted in the repertoires of

basement membrane degradhg enzymes produceci by the two subünes. H-59 cells were

found to secrete higher levels of the 72 kDa collagenase (MMPZ, gelatiaase A) and

cathepsin L whereas M-27 cells were found to express higher levels of the urokiaax type

plasminogen activator @PA) and cathepsin B, suggesting that invasion by these ceiis is

differentially regdated (392).

These finduigs are swnmarized in table 4-1.

Table 4-1.

3LL Meîastasis Adhesion Expression of Matrix ûegrading Proteinases

Subline Liwr Lung LN' LN ~ e p ? MMP-2 m p C 6 -p. L "PA*

a LN, lymph node b- Hep., he~atoc~te c. Cathep., cathepsin d. uPA, urokinase plasmmogen activator

(C'.L.CER R E S l A R C l I -54. 172-377 . JJb 15. 1-1

Paracrine Growth Stimulation by Hepatocyte-derived Insulin-like Growth Factor-1: A Regulatory Mechanism for Carcinoma Cells Metastatic to the ~ iver '

f Li Long, John Nip, and Pnina ~rodt' Lh-parrmcn:.~ of Sur.rcn /L. L.. J 'V.. P. 8 / und Qnccdq~~. lP B. f. .W&N L:ruwrsiiv and he Rmd Vkmria Hmptml /P. B.1. \fon!reuI. Quebec H3.4 1.44. C m h

Tumor H-59 is a sublinc o t tbc Lewis lung arcimoma which is highly and pmfercntially metastatic to the liver. We uxd tbb atdwmm m o d to investigate the rok of paracriac growth regulation by liverderived factors in this organ-selective pattern of metastasis, We observed that scnrm-free medium conditioatd by primary cultures of mouse bepato- c y t a was highly and specifialty mitogcaic lor H-59 crlls but bad littk etYect oa the proliferation of a second sublinc. Le.. carcinoma M-27. which is metastatic only to the lung. This mitogenic activity was bepatmtc- specific and could bc blocked o r depkttd by a monoclod antibody to insulin-like growth factor 1 tIGF-I). IGF-t cwld in turn be detected in hepatqtc conditioned medium by Ihe Western blot assay. and whea added to xrumdeprivcd cells, IGF-1 could stimuhte the proliferatioa of H-19 but not M-27 cells, Furthermote. when expressioa of the IGF-I receptor was analyzed by the Sorthcra Mot assay. we l i ~ ~ n d hat H-59 cells expressed signifhxatly higber kv& of mRWA manscripts eacodiag IGF-1 receptor. A ligand binding assay revealed that the number o f IGF-I binding sites on H-19 ctlls was 3.4-fold higher than that on 51-27 cells. The d t s identify ICF-L as the growth factor mediating tbe p d f e m t i v t effect of hepatocyte conditioned medium and suggest that porrcrinc growth stimulation by hcpotocytc-derived IGF-1 is a potential mechanism of selection in the process of liver colonization by these carcinoma cells.

The mechanisms u hich determine the hilit! of cancer celIs to t o m metastases in specific organ sites are diverse. Evidence derived from clinical and experimental studies suggests rhx. in addition to mcchan- i d factors such as blood flow and lymph dminag. specific intenc- rions between the cancer cells and the ce1Iuhr and cxtracellular elements of a target o r p site 3re involved (1-5). These host-tumor interactions include cellceIl and celltxtnccllular matrix adhesion (6-10) and growth regulation by orzan sire-derived or -activated growth factor(s) ( 1 1-13].

To study the host-rumor interactions regulating liver metastases formation. we developed an animal model based on two sublines of the murine Lewis lung carcinoma which are metastatic. one prefer- entially to the liver (carcinoma H-59) and rhe other specifically to the lung (carcinoma M-27; Refs. 1 1 and 15). We investigated the role of pancrinc faaors in regulating the ability of these tumor cells to form liver mctastues. HCM3 was found to be mitogenic for H-59 but not 31-17 cells. raising the possibility chat a hepsroqte-derived growth f a c t ~ ~ r (or factors) can regulate hepatic dissemination in this model.

To identiiy the hepatocytederived factor(s) and the mechanisms underlying the divergent responses of the two sublines. a combination

ZCCCIVCJ 3.3 OJ. ;~~xepfcu 5. 10194. Thc costs of publication of this anicle wcrc dcfnycd In pan bv the payment of pagc

charges. This rrrticlc mmt rhercfnrc be hercby marked udwrrzmcnr in xcord3ncc wth IS L'S.C. Section 1754 wlcly to indiafc t h a fact. . ' This study was supprtcd bv 3 grant from rhc M d i d R e d Council of Cam&. 'To whom requests for reprints a u l d be addrrucd at Deprrmcnt of Surgery.

Diklsion of Surgial Research. %lcGill University. Donncr Btdg. 740 Dr. Prniicld Awe., Munxc~l . Quebec HjA i.44. Canada.

' The abbreviations used arc: HC.U heptocytc conditioned mcdiumr IGF. insulin-likc g r w t h factor: IGFBP. insalin-likc growlh factor binding pmrcin: C!bf. conditioned mcdk PDGF. piatclet-derivcd growth factor. TGF. rransfoming p w h hcror: W. mono- dons1 antibody: cDKA complementary DNAt SF. serum I=: BSA. buvine =rum dlrurn~n. SDS. M i u r n dodcql culfatc: SDSPAGE SDS-palyacryhide gel clcam- phore\i\: EGF. epidermal growth haor: HGF, heproqte growth fsaor.

of immunochzrnistry. [isand binding assays. and Nonhcrn blot a n d - yses were w d . We show here that IGF-L is the major mitogenic factor for H-59 celIs in HCM and that differences in the IGF-1 receptor density on the variant cell lines and possibly in the synthesis of IGFBPs a n account for thcir J ivcrgnt responses to this growth factor-

Cell Lines. Tumors H-59 and M-17 were established from hepatic and lung mecrtskxs of the p m n t line 3LL rapcctively f la t Thc tumors were main- tained m vivo by s . ~ . implanntton o r lung ( ,M-17) or liver r H-59) rnera<txxs derived from tumor-bearing mice into new recipient animals. In vitro mono- layer cultures of the tumors were maintained 3s detailed elsewhere I ~ I . To prepare tumor cell CM. subconflucnl culturcs were washed rwia with SF- RPSfZ IdM and then incubated with the SF medium for J8 h- The media werc tollccrerf. aliquoted and stared at -2D°C until used.

Reagents. Human recombinant [GF-1. IGF-2. PDGFs rU and BB. EGF. TGF-a and TGF-8. and the antibodies to these f;rctors. LL. J m o w M.4b to human [GF-I. a rabbit antrxrum to bovine IGFBPZ 3 nbbit .inriserum to EGF. and 3 goat antiserum to human PGDF-AB. were purchased f n m C'BI t L l i e Placid. SY). The bovine HGF was a kind gift from Dr. M. Park (Departmat of Oncology. Royal Victoria Hospi~aI. Sfontreal. Quebec. Canada), The moitx MAb to human TGF-83 was from Oncogene Science ~Uniondalc. SY,. Al- kalinc phosphatrrse-conjuga~ed anti-mom. rat- and nbbit antibodirs ucre from B[O-ChV Sc-isntific i51Lssis.s1ug~ Ontario. Canada). cDS.4 1t.r rbs human [GF-1 receptor was tibt~ined from the .4merian Tpc Culture Cdlec- tion ~Rockvilk. %ID). and an otigonucleotide probe for IYS rRV.4 was a kind eift from Dr. 0. Blaschuk (Division of Urology. Royal Victoria Hospiul. .Montreal. Quebec). Protein .A Ssphamsc C L l B was from Pharmacia Biotech. Inc. (Bait d'Urfe. Quekc. Canada). immobilized P r o n w from Pierce :Rock- ford. IL). m o w albumin from Sigma Chemiul Co. (St. Louis. 510). and bovine x rum albumin from Boshringer Slannheirn Canada ( L i b s l . Oucbcs. Canada t. [$'P)ATP t 3 0 0 Ci mmol). [a"PldCTP (30(lI) C i mmol). ~ n d ['H]thymidine (2-0 fummol) wcrc from Du Pont Canada (Sf'ksisiup. On- r;lrio. Canada). "I-labeled IGF-1 (2IX)c) C i mmol) was obtained fmm .-er- sham Canada (Oakvillc, Ontario. Canada).

Preparation o f Mouse Hepotqtcs and Lung T i c CM. Ci-BLo fcmale mice. 7-12 weeks old (Charles River Canada. St. Consrant. Quebec. Canada). werc u x d as the wurce of organs. Pnman cultures of hcpatoc~tcs wcre prepared by the method we described previously (15). The hepatocyte monolayers were washed wic t with SF-RPMI 1MO 48-72 h after plating and then werr cultured with SF-RPVI IW) for an additionai 4.S h. HC51 s3s

colleaed. aIiquotrd. and stored at -20°C for future use. The lung Clcf uas prepared using the method described by U'halrn and Sharif ( 16). Bnefl>. rfic l u n g wcrc minced to yield 1- to 3-mm' fralynenls. and the fragments ucre washed several times with phosphate-buffered saline. placed in culture dishes containing SF-RPMI 1640 ( 1 mL mg tissue). and incubated at 37°C for 14 -ib h. The medium was colkaed and centrifuged to remove tissue debris. Lrber- organ culture CM was prepared in a similar manner. To concentrate the CXf. they were lyophilizcd in 3 Hf3OfRAP CT L 10 Iyophilizcr (Heto Hi@ Technology. Wndinavia. Denmark). Prior to use. the lyophilhtc was rccon- stitured in 0.1 st acetic acid and dialyzed for 12 b at J'C against 0.0 1 M acetic acid.

Tumor Cell Proliferation Assay. Semiconfluent rvitures ot H-59 and M-27 celk gmwn in RPMI 1640 with 10% fetal calf serum were washcd twice with SF-RPMI 1640 and cultured in SF-medium for 24 IL h'lhc cells were dispersed with phosphate-buffed d i n e - E D T h and 2 x I@ cclWwel1 wcre seeded onto 96well polystyrene plates (Falcon) together with the growth factors or CSl and with or without antibodies s specified. Following a 5-l-h

incubation. the cclls were pulsed with 0.1 pCiml of ['Hlthymidinc for 18 b Thc cclls uen: lyscd by rcpeatcd i r c a i n g and rhawing. and the cc11 lymtcs wcre hamesrcd onto p p c r filtcr -inp the Micromitic 146 h a m a t e r ( P x k a r d Insinimenr Cwnpany. Inc.. Xleriden. CT). ILHJThymidine incwrpimtion w;r\

monitorcd usinp the Beckman LS 8000 liquid scintilhtion counter (Beckrnan Imtruments. lnc.. Fullrnon. CA).

~ M S C Digestioa of HCM. HCM was incubaicd wirh irnrnobilitcd Pro- nase (Pierce. Rockford. IL) for 12 h at 3PC. The final con~rn tmt ion of the enzyme was c~lculated to pive an enrymcisubstntc-protcin n t i o of 1: LOO. The Prcinase sas rcmoved by ccntriiugation at IIWWI .r. S. Conrrul H m 1 sÿmplcï u c r r i n ~ u t u t e d iit j7'C for 12 h without Pronac.

Rcmoval of IGF-I and PDGF from HCM. Niquors of HCM wcrc incvbarcd with a rnouse M A b to IGF-1 or a goar polyclond antibody 10 PDGF-AB ( 1-20 pg ml) for 12- l h h at J'C with agitarion. Tc) m c h aliquot. rabhit .mi-mouse lgG or rrihbit ami-goat IgG u t u n t d Proiein A-Scphamsc CL4B beads wert added for an additional incubation of 12-16 h at 4-c (- 100 p l k a d s ml HCV). Prntrin .a k a d s wcrc rcmovcd by centrifugation. ControI HCM w p l c s wcrc trtatrd in an identical rnanncr but withour thc addition of the t in t anribody -

Western Bloc A n a l y i s of Crowth Factors and tGFBP 2 ia rhc CM- wcsicm blot anal!& w s crrried out usine cs~btished p m c e d u m (17. 12%). Conccntrrirrd a m p l e s of mcdia conditioncd by hcpaiocyta. H-59 and 51-27 ccils. o r lung o r g m frrigmcnrs wcrc loadcd onto 15% p o l y a ~ l a m i d c gels; at 150 r- prorrin per Ianr: and wprinred by elearuphorcsk undcr nonreducïng condiuons using an LKB minigel systcm (7050 rnidgcgct Pb;innacia LKB Biotcch Inc.. Baie d'lfrfc. Que). Folluwing electrophoresis, the proteins wcrc rrrinsftrred onto nitrocellulose using a T n w B I o t apparatus (Bio-Rad Labo- rritarics. Richmond CA). The blols wcrr p m k d with mtibociics CO p w r h factors and io IGfSPt (LW). Following a 12-h incubation with the prima- mtihoriics. blors u e r e \*shed and incubaied with fiie appropriate alkaline phosphar3scconju~~lcd second mtibodics (6IO.C.'; Scieririfici. The! were dcvcloped with the substme nitro b h e tttr;uolium5-brorno-k-hlo~>in- dolyl-phospharc as we dcscribcd prcvioUly { 19).

Watcrn Ligand Blotting .Asa? for 1GF Binding Proteins, The asmu was performed asentially JS d m - b e d bu Holly r.r UL (3)). Brisfly. samplcs of CM ucrc: Irudrd onto LUC; p l j a q l a m i d c gels nt J srinccntmtion of 150 p g protein lane and ricpar;itcd eltarophoretic.il~ undcr non-rcducing conditionx The separaicd proteins uerc t n m k r r c d onto the n i t r ~ e l l u l o s c tilters ;L..

describeci above. The filters werr blocked with Tris bufirred saline containing CCr skirn rnilk. 3 5 BSA. 11.15 Tuecn-20 and incubard obemight al 4'C with ["IIlGF-1 adjusted ro a soncrnrnrion of 4 7 10' cpm ml of blocking buffer with or m-ithout 3W ng colci IGF-1. Bands cormponding to the [GF-1 binding proteins uerc derecled using auiomdiography. h x r dcmitomecry with 3 LKB Bromma Cltro-imn SL Enhancd h r ïknsitomercr sas u x c i to quantitate the rçlaii\e intensitj o r rhr. bands.

N o n h e r n Biot -4nalysis. Cdlular RVA &ris cxtrriacd irom W-59 and 51-'7 c c l b b> the phcnol-chloroform rnsrhori of Chomcz'nski and Sacchi (21). The R S X ,pccies wcrc r~ .wl \cd bu slcctruphlires~r in 1'; g a r u x $1'. iuni;iininp - 3 . 3 fomri tdeh~de 1221. Approximattly 50 pg of total RSA wsrc loaded onto sach trine. The tmaionateci RNA species ncre tnnsfenrd onto charecd n r l m m r r n b r ~ n r s (GencStxen Plus: Ncw Engiand Nuclear Rcxarch Products. Bosron. MA) b' the L-apillriry blocring rnethd. .A S - k b EÇURI resiriaion fr;igrncni ui the human IGF-i rrsrptor CDS.\ uzcd a, a hjbndiz;itii>n probe attcr if UL+ rdi tkibelrd by ihe rrindorn primer methocl of Fsinbrrg and \ argrl?;rrin ( 3 1 . The blocs wcrc incubrited uith the r~diolahclcd prolx for ' 4 h ai 37'C. uÿshed. and subjectcd to a u t o r ~ d i o p p h y . The relative amountj of the mRSA tramcripis uerc analyzed by laser dcnsitometry usine an LM3 Bromrna Cltrowan !U cnhanced laser densitometer and normalizcd rcbrivc 10

rhr 18s rRSX. Binding of '31-CGF-l to Xloadaycr Cultum olTumor Cdls. T o quan-

rirais rhe binding of IGF-1 to the turnor cells. the rncthod dcscribed by Phillips c.r ul. (24) w;is u x d . Bricfly. H-59 and M-27 e l l s wrre culnrrcd with RPMI IMObfecal n l f wrurn in 2 l w c l l plates for 1-3 daus. The culture medium w u rrmoved and rcplaced with SF-RPMI 1630 conwining 1 msfml 8SA. Binding ;LS.W)\ nere carricd uut 12 h bter. To each ..vcll. SF-RPW 1oJO containing S-1500 PM of l"I-labekd IGF-1. 1 m y m l BSA. and 1 pgiml leupcptin wcre addcd. with or without p d c d concenuadons of unlabelcd IGF-1 for a 1-h incubation at 37°C. Thc cells wcre cinxd twicr with icecold binding medium and wlubilized in (1.01 \ SaOH mnain ing 1). 1 5 Triton S-l(Wi and (1.15 SOS.

Cc11 numùcs/wclI at the cime of ihc sisay wcrc &temincd h m triplicatc conirol wells which wcre rnanipuhrcd in rhe samc manner. An diquot from c x h wcll mi rcrnovcd, and the mdioactivity urar monitoml in an LKB gamma ~xwntsr. The d l \ . i a t i o n cumiant ~ n d the nurnkr of IGF-1 binding sites f a a c h d l lins w m c a l c u l ~ e d using the Li@ p m p u n (3. 3)-

To test whether l iver parcnchymal cells produccd factors which ~r,ulJ mcdulatc carcinoma ceIl grawth. wc culrurrd ucrum-\tan.cd H-59 and M-17 cells in SF medium conditioned bu HCM. Hrpatic endothdial celis and fibroblasts were used as conrrok. Rrsults of a repmentative expenment are shown in Fig. Ia. HCM was highly mitogcnic for H-59 but not 31-27 cclls. incrcz~ing ['Hlthumidinc uptdce by 1 1-fold and resulting in an S-fold incrrasc: in ce11 number. In a ioal o f five additional expenrnents. the increase in ['Hlthymidine uptrikc in rssponx to HCM nnged €rom 6.2- to 27-1-fold for H-59 ~rlls but was negligible for M-17 cells. Media conditioned by fibro- btasts md rndothslial c e k had no effçct on proiifrntion of citbrr turnor Iine. whereas Iung or-- c u f t u r c CM stimuhtd both cclf types [Fi& 1dL

Scsvenl Iiverderîved polypeptide growth factors have previousiy brrn idrntifird. They include IGF- 1 (27-30). IGF-2 (3 1 ). TGF-a(S2). PDGF (33). and HGF (34)- We tested the cffects of these factors on the proliftmtion of H-59 crlls and compared them ro the sffect of

HCM. Results of 3 representative experiment are wmmarized in Fig. Ib. IGF-1 stirnuiated DKX synthesis in H-59 but not 34-27 cells. This effect was dose dependent (Fig. 3, reaching a 4-5-fold increase rclativr to control cell3 at a dose of 0.h n\l ( 5 n s mi) IGF-I. In tive adifitir~nal evpenments. the incrcas in [ '~lthymidine uptake in rc- sponse to 0.6 nsc IGF-l ranged from 2-1- to 4.6-fold for H-59 cells and u a s ne_gligible for 51-27 cells. Other growth factors were tested at concentrations r~ng ing from 0.001-13 nat. IGF-I and PDGF-aa had a minor stirnulaton effect on H-59 cells. whereas all other faaors testrd h d either no effect ur 3 minor prolifer~tive effect on both H-54 and 51-2' cells. Representative results obtained with J concentration of 0.h nst of these factors are shown in Fig. 1b.

Initial chamcterization of the HCM factor mediatin2 the mitogenic cffect was provided hy subjecting the HCM to Pronasr digestion or to a temperature of IoCIcC. These treatments reduced the prolifentivc effect of HCM by 70 and 9 Y 5 . respectively. indimtins that 3 protein factor u a s intolved (data not shown).

To identify the growth factorts) mediatins the stirnulatop cffea of HC\1. nrutr~lizing M.47 were subsequently used. The results shown in Fig. 3a dsrnon.;tr:itt: :i significant and specific inhibition nf !he mitoernic effect with MAb to IGF-1. The mito_penic activip could be depleted from the medium when antibodies to IGF-1 were u x d in combination u-ith immobilized Protein A This effect was dose de- pendent and w= not seen when a polyclonal antibody to PDGF-AB ubrd as a control or only protein A beads u e r s added (Fig. 3b). Depletion t ) t IGF-1 from lung CS1 also resulted in a decrease in the \timulator? effect o i the medium o n H-59 cells. reducing it bv a factor of 35. but it had no effect on stimulation of Xf-27 cells (not shown).

When the Western blot assay was subsequently w d to detect [GF- I in HCM w i!h an anti-IGF- 1 MAb. two specific bands of :\I, 4S.OW and 2S.(HX) were seen (Fiy. 4). suggesting that IGF-1 in the CM was present in a complex with other. h i e e r molecular weight proteins. IGF-L was also detected in lung C M .but no IGF-1 could be detected in media conditioned by either of the nvo tumors.

The objective of subsequent experiments was to elucidate the factofls) C respunsihle for ihc di f fc~nce in the response of the two sublines to [GF-1. Two possible rncchmkrns were investigated: (a) differences in IGF-I receptor ( IGF-I R) expression: and (6) differences in the produc- [ion of IGF-I binding proteins. A lipnd-bindins assag wa5 w d to

measure receptor &nsity and the bimling affinity of IGF-I to the tumor cells. RcsuIts in Fig. 5 show that the number of [GF-I receptols on M-27 cells (b. 937 binding sitedcell) was significantly lower than that on H-59 eIIs (ct. 3200 binding sitescsll). In addition. the binding affinity of ICF- I to M-27 cells (K, = 950 pw) was found to be si_enificintly lower than that to H-59 cells (K, = 193 pv).

Funher confirmation for the difference in IGF-IR expression in these variant cell lines was obtained from Nonhem blot assays. An I I-kb mRNA transcript corresponding to the IGF-I R could he de- tected in total RNX cxtmctc of h ~ t h turnon. Htw i.t er- ~ n a l ~ s i 3 o l thr bands by laser densitorneuy revealed that H-59 cclls expressed 5-fold more IGF- IR mRNA than M-27 cells (Fig. 3c).

IGFBPs are a g o u p of polyeptidcs which a n bind IGF-I and augment or inhibit ifs function. We investigated the pussihiIity that the difference in the potentids of H-59 and M-27 cells to respond to IGF-I may also be related to differences in the ability of the.% c ~ l l s to produce IGFBPs and bind IGF-I from HCM- Production of IGFBPs was andi~_vted by the Western li~and-binding assay using CM derived from the tumor cells o r from liver and lung or_ean cultures and

0 1 2 3 4 5 7 8 9 1 0 t 1 1 2 1 3 Prorain concmntration of HCM (pglmL)

Fig. 3. Inhchlt~on and Jeplctim of rhc rniropn~s c? t s t ur HCM h? a m~~nm-lmal mibody to IGF-1. 4 earn-starved H-59 cells were inrruhtcd iar 7 h with dilfehlnt cuncenuarions o f H a in (hc abscncc (3 or presence of m u M i a to IGF-I ( C ) or EGF ( 4 ). b. diquoa of HCM were incubated with lCWb m tGF-I md nbbif anti-mouse I& antihdv-utuntcrl pmrein A-Scphrrox heads i - I lU) pL ml; t Thc k.rr3& acre rcrn~ircrl by centrifugation. A pol~clorwl antibody TO PDGF w s uxd ;LI ;I cunuul and Jcplctd uith nbbit mti%wat IgG anribody~runtcd protein A-Sephmxe kds (m. Scrurn-scmed H-59 cclls sere culturtd u-ith COOMI or antibody treated H-I for 72 h The raults ;rrc

m a n of niplic=rc wmpla: Iwrx SD. They rrr c n p m ~ the pcmnnge ~rimulation rchtcvc to H - 5 s cdts incubated wth f;CM pretrcarcd u ~ r h Pnmxn .A onl?.

'"1-labclcd IGF-1. Two binding proteins of .c1, 55.000 and 24.000. 6 7 corrnponding to binding pmtcinr iGFBP3 (M, S 5 . m ) and IGFBPS

or I (M, 21.0003. wcre detected in CM h m the tumor's and organ culturcs {Fig. 6a). A third binding protan w u specificdly recoynized by sn antibody to IGFBP2 (Fio. hhl. H-59 cclls producrd si_pnifiantly mort IGFBP-2 than M-27 cells (a 4-kild differenco). Intercstingly. the

C concentration of IGFBE in luns CM was signifirxntly higher than in cither liver CM or in HCM.

Fig 4 Dcimion of IGF-l in H a and ui Iung CM b) W ~ m i bIot mdysis. C-M pmicim 11-9 pglanc) uert w e d by SDS-PAGE tr;insfmcd onro nitnxrllulox rnemhrsnfi. m d incub~lcd wirh mubody IO IGF-1 t l petni). &ne 1. 5 0 op human rltiF-l Ldncr 2. -;. J. md 5. mcdta conditioncd by hcpaoqia . lung iiuuc. H-Cu. ~ f l d

)cf-:' :CIL. rcyxai%cl% Lunes o and 7. HCSt and lune CM p M w i h a ~cunrml munnc SfAb ri, ln r n c b a n : proretn. Lzfr pic ions cri molcculu uci@r

DISCUSSION

Tbc cellular and molccular interactions which regulate the potential of tumor crlIs to rnctrist=izc IO different orgin bites are diverse and both tumor and organ specific. Evidrncr: derived from several exper- imentai models suggests chat paracrine stimulation of tumor cells by or_eandrrived growth factors is one mechanism which determines the target organ prefcrence of disseminating cancer cetis (2-5. 10.1 1. 16).

Usine two carcinoma sublines with distinct patterns of dissemina- tion. we dcmonstratc here that the ability of tumor H-59 celis to colonize to the Iivrr was ascciatcd with a signifrcmt increase in their prolifenrive response to hepatoqtederived IGF-1. We iound thar thcsc tumor celIs cnpressed increased Ievels of IGF- 1 R (as compared to M-27 cclls) and produccd at l em nvo IGF-binding proteins. yct they did not secrete detectable lsvcls of IGF-1. The lack of IGF-1 prociuctioa was alm confirmed by a Nonhem bloi m r i y which t i k d to detect the bands corresponding to IGF-1 mnscrîpts in RNA ex- tram of H-59 (not shown). Thus. dthough these rurnor crlls have the cripabiIity to respond IO the growth hctor. they appear to be dependent on an exogenous source of [GF-1 ro triggrr a prolifentivz responsc. This dependence on a pancxint source rendes the liver a favorable environment for their growth.

51-27 cells aIso express the recrptor for IGF- 1 and produce dercct- cible lsvels of [GF-1 hinding protriins. Thrir failure to rrspund ro IGF-1 may be related to the Iow lrvel of IGF- 1 R expression and'or the significantly deaeasrd binding affinity of IGF-1. While rhc mscha-

Fig. 5. Scatchard Yialysis of IGF-f b i n d h and Nonhcm blot -y for IGF-IR rnR'iA tmncnp exprcsscd by tumor cells. Differcni coacarr;ilions of '31-Gf-1 acre addcd 10

26). Toul RNA (50 w l a n c ) dcrivcd from thc w o c c U liaes was maiyzed using z 2-7-kb IGF-IR cDNA as bybnd~rion p r o k (c). The 28s and 18s nbmonul RN& wcrc uxd as H-59 (a) and M-27 ( b ) monolayers i t 37.C for 1 b The d i ï i i o n CO-i and the n u m k of IGF-1 biading s i i a for cJch cc11 linc wcrc d c u h i d uYng the Ligsnd p m (5.

sizc msrkca. Thc r a u i t s of hybridiition of the u m c filier with the 18s oligoaudeotidc pmbt ut sbom at Ihe m m . Bands were m n c d using the LKB Bmmm U ~ ~ ~ S C M XL cnhanccd laser dcnsilomcicr. and the diffcrtmx in the qwntq of m&SA i w n p t s w s dcuhtcd darive to the 1s smbrds. The inrtnsiry of i k I I - k b band dcrivcd from H-59 celb =as 5-fold higher t b n i h i denved fmm M-27 alis.

3735

nism responsible for the low-ered affinity is not yet cfear. it could concriv~bly be due to posttranslational modifitxtions in the recrptor. Wr also crinnot exclude the possibility that defects in signaling rivents downstrcam of the IGF- 1 AGF-1R interaction contributs ru the lack of rnito_eenic response by M-27 cells.

Taken to_eether. t h s e results sugxst t h a ~ in the prexnt model. the divergent ptentials or' the urmoma ce& to memusire to the liver is replated by theu ability to respond hvorably to hepatoqtederivcd IGF- 1. Thus. our resuits provide rhc: first line oicvidence that IGF- t ma); rcplare wncer cell dksemïnation to the Iiver. In th& respec~ it is relevmt to note recent reports t h t colorertaI carcinoma tells. for which the Liver is p w n l l y a prim- site of metasusis ui vko. were similady stimulated tn \in-O in responx to 0.WS63 nw IGF-1 (3437).

The role of IGFE3Ps in genenl (38-11) and in our mode1 in particular is nor !et full? understood. In preliminary esperimrnrs, we found rhat antibodies to IGFBPZ enhancrd the prolifer~tive rtsponsr of H-59 crlls to HCM (cirita not shown). This is consisrent with results rrpunrd by other investigstors (12) and may indicrite that IGFBPl could play a nezative re_eulrtton. r ok in IGF-1 function. rUternatively. antibody binding ma'; result in conformationaI changes which in- c reax the affinity of the IGF-1:IGFBP complex for the receptor. or it ma! incrcrtsc the stabilit- of the complex. .As rintibodies rractive with murine IGFBPs 1. 3. and 5 rire not prexntl); rivailable. their function in our modrl mnnot br casil? drtrrmined. Howrver. the hi* Ievel of H-59 prolifention in responx to HCM suggests that the net contri- bution of the binding proteim prewnt in this medium is to suppon the mitognic rfftxt of IGF- 1.

We found char lung CM. HChf. and IGF-1 wrre 311 xnitogenic for H-5'1 cells. but only lung CM stimu1arrd the p w t h of hi-27 crlls r Fig. 1). This suggtsts thar. in lung ClIf. a factor (or factor';) other than IGF-1 ma' hc prcsenr which crin prornore the prolifention of hl-27 cells. In r-iw, this factor ma! contributr ro the seleaivr jrowth of metastatic hl-27 cells in the h g . On the other hmd. the preferentia1 metaïtasis of H-59 cells to the liver. despite the prrscncr of high Ievels of IGF-1 in the lune (Ref. 23: Fig. 3) could be resulated by addirional fricton such as specific cei l te i i intcnctions (19). The findinz of high levels of IGF-1 in lune CM may also rxplain the abilit? of H-59 cells to grow in this organ following the inoculation of high doses of tumor crlls directly into the pulmonary vasculature. as we previously repmed (14). It is a h possible that undcr these conditions autocrine mechanisrns ma) promotri tumor ceil protifen- tion in the lung independentl'; of paracrine growth factors.

Our resulrs raise the possibiliry that the IGF-I receptor couid provide a rrirgrt for specific thenpeutic intervention aimed at inhib-

iting the purfr of hepatic metastriscs. Rectnt studies have shown thst a monoclonal antibody to the human IGF-I receptor could inhibit the growth of the human breast carcinoma ceil line .MCF-7 in rirro (43). This mtibody also inhibitrd human melanoma cri1 _murth in culture and in athymic micc (44). uthibition of nirnor ccll growth could also k schieved with IGF-f a n a I a p s and following tnnsfection of mnlignant ceils with plasmici vectors expressing [GF-1 or IGF-1R cDNAs in the amisense orientation (45-49). For carcinomas which are respnsive to IGF-I and metastatic to the liver. thex and similar approaches coouid lead to the devdopment of new stntegics for the prevention and ueatment of liver metastases.

REFERENCES

anrinoms v-JnJnt H-Sir idcntiiin s plwm mcmbnnc prurcin uith Jppucnr rclc- iwcc ir> I>mpn n d c dheicn and mcusc*i> C~nccr RCL 50- I%i;-l+S. : UUO. Bror l~ P. Adhaion rnciunisms tn lympiuiis mcasfs i s . C w c r %fcms[;i,is Rm.. f«: LM' 1991. Pauli. B. C.. Johnson. R C.. Widom. J.. md Chan+ C-F. Endi~thclial ~ ~ 1 1 rdhc- \ion molsolles snd rhcir rolc in o r y n prcfcrcncc of rncr;ÿmr\. TrcnLi GLyma. GIymc~hnol, 4: 405-414. LW- Pauli. B. C.. Au-auin-Vocs K G.. U-Sabbui. M. IL. Johnson. R C.. and Hmmcr. D. A Orgin-preic~cncr: or mcwstum. Cancer Slemt;lsis Rcv.. 9: I Ï t l S U . LW. Rusamo. O., LL>rcnzoni, P, and Burgcr. Y. M. The wle o f horh spccific cellular adhoion and grnulfi pmmorion in livcr ~vlonintion by fs cmbryon~l &nom;\ alk h[nt S. Cancer. J8: 45û-456. 1991. Rdimky. R. Gmuth factors and rhcir rctcptors in rncu4iJsis. Semin. Cancer B I I L 2: lbq-[y. [ W I . Saiio. S.. GWvJni. R. snd Fïdlcr. 1. J . Corrchiion kwccn the ur vizro intenaion o f -or cclls with an o r g m cnvironmcnt and mcr;ismtic bcbvior m vnv. Invasion .U~W~SU 2 I b - 3 . 1 9 s . Honk. L. Darling. D. L. and Tmn. D . b l y % i s of or- \pcofic cffccri un

mcu~otic ntmor formation by sudia III rim. J. NaiL Currr lm. 76: 913si-t. l w s .

14. B m d ~ P. ChurcicMiion of nro highly merasntic vaciau of the Lewis lung c~nmwrna wih diffcrcnt orpn cpecikitics. Cancxf Re.. 46: 342-:LUI. 19%.

15. Undi. P Sclccrion nf J h@ly mctsatic l i ux~ .bnu inp wbpopulaimn of Lrwr5 lung amnoma variani H-59 using murine bcpoioc)l<e m o c i d a y m Clin. Erp. \ f c - ias~~ ' 525-5.W. IWQ.

lh. Uhalcn. G.. ~ n d Shini. S. b l l y incrcrsai mcasoiic cffecncy a r fusan for prtfcrcniial rnctasasis of wlid tumm io lymph nodu Ann. Su+ 21% 166-171. I Q Q I

1, Twbin. H.. and Codon. J. lmmunoblotting and dot i rnmunobind inmnt e t u s and ourlook. J. Immunol Mcthods. 71.- SL-L-;U). 1981.

IS Touhin. H.. Siachciin. T. ~ n d Gordon. 1. Ek7~ph0rCt i~ innsfer of prnlein.. frum

Loss of the Metastatic Phenotype in Murine Carcinoma Cells Expressing an Antisense RNA to the Insulin-like Growth Factor ~eceptor'

Li Long, Raphael Rubin. Renato Baserga. and Pnina ~rod?

ï h e abitity of malignant cells to form mc(rrtnra in ueoiiQn r i ta remains a major obstacle to the curative trcatment d cancer. P r e v i d y . w e ideatified type I insulin-like growth faciof t [CF-1 8s 8 purrine mitogen fat highly mecastatic munnc arcinomn, H-59 c c k Htre the rok of IGF-1 and its rcceptor t IGF-LR) in w ~ b t u d e r iavatiltptd using H-19 relis transfected 4th a p&dd vcctor upm KGF-IR cDSA in the anthense orientation. The t d e c ï a n t s bod a markedly reâuced expression o f [CF-IR and lost the ability to respoad ro IGF-t in vitro. When injected in vivo. cicber d i d y inîo tbe m i c r o v a s c u l a ~ of tbe Lvtr or lung (experimcnlnl mdashdsl or s.c to rllow tbt go* of primary local tumors t spontanmus melpnnsist. tbcic ctlls did mt give rW CO any mefastases under conditions which allowcd wild-type or convol transfectants to form multipk heprtic and pubmmy +bc results demonstrate that the [CF-IR can piay a criticai role in the reffu- htion of carcinoma metastasis.

Introduction

Despite major advances in our understanding of the molecular mechanisms underlying rnalignant transformation, progess in the clinical manasement of cancer remains lirnited. This is due in large pan ru the lirnitec! success of conventional thrnp? in the rrearment of second- metastatic lesions (1). Cancer metastasis is a complex rnuhistcp process involving detachmen; of mmor ceils from the pri- ma- site. invasion of and migration through blood or lymph vessels. c'ctrrivasation into distant organs. and ceil proliferau'on in the target orgrin in response to autocrine andior louil:; relcrasctd growth factors ( 2 . 3). Sumcrous studies have shown chat the acquisition of a malig- nant phrnotype is associated with altered responses to various growth factors due to changes in the expression of growh factor receptors andor the altered regularion of grouzh factor-induced sipal tnns- ducrion mechanisms (4).

Evidence is npidly accumularing that I G F - I ~ and IGF-IR play a crucial role in malignant transformation (5). Recent studies have shoun chat antiscnse expression piasmids to IGF-l(6) or the receptor ('1 and anrihodies to the [GF-IR ( Y ) could inhibit turnorigenicit). In sorne rnodrls. this was artrïbuted, at Iras paniall~. to rnoduIation of curnor immunogenicity (6). The role of IGF-1R and i ls ligand in the regulation and maintenance of the metastatic phenotype has not. however. been investigated.

Tumor H-59 is s highly metastatic variant of the iewis lung carcinoma (9. 10). Previously. we have shown that the potencial for

Rcaived 1: 20i9-k amprcd 1; 19.95. Thc cosu of publicauon of this Ytictc wcrc dcfnyed in p ~ r by k pymcnt of page

charges. ni6 article mus chenfort bc hcrcby mutcd uhenircmrnr in accordancc 6th l b U.S.C. Seaion 1% wlclv IO indicilc this f 3a

Thb work was supponcd by a gnnc €rom the Medicd Rts tmh Couml of Chuda (ro P. B.) and a i s by Zm( G m u A.A-0123 and IU-07309 (to R R) and CA-53484 (rn F. B.) - To wham rcquats for reprints sbauld bc JddrtsKd

' Thc ~ b b r c u i ~ t i o ~ u u d JE: IGF-1. type 1 insulin-like grouth haoc IGF-IR [CF-1 rrccptor. DSlSO. dimethyi iulfoxidc; HCM. heptocyrc conditionni medium. inm- \plcnic; STM. w u m frcc medium

hepatic metastasis in this mode1 correiated wilh increased responsive- ness to hepatic IGF-1. which w s in turn due to increased expression of the IGF-1 R (1 1). T o study the role of IGF-IR in H-59 growth and merastasis in vivo. tixe mmor cclis were transfcctcd with a vector in which IGF-1R cDNA is constitutively exprcssed in the antisense or (as control) sense orientations relative to the SVJO promoter. We show here that the antisense uansfecrants lost the abiiity to respond to IGF-1 and hepatocyteconditioned medium in virro and failed to give r ix to memtases in vivo.

Ceîi Lints. Tumor H-59 was cstablishcd €rom a hepatic metastases of the parent Iinc 3 U (9). The m o r *-as maintahcd in L-~L-O by s.c. ïmplanmion of liser metastasci dcrived frorn tumor-k3nng mice inro neu recipicnt mimals. In \.irro monolayer culnirrs of the turnor were mainnined in RPMI cunuining 5% FCS aç dctailtd e k w h e r e (3-

Constn~ctioa of IGF-IR Plnzmids. .4n .WI-.YhoI frqtnent correspond- iog ro 1-339 base pairs of the IGF-1 recepror cDNA ( 131 was lïgatcd into the .Ybat-BmHI site of the CVN veffor (14) in thc xnsc or sntiscnse oricnmions relative to the SV40 cari! pmrnoter o n e . The vector 3lw fiintains :hr dihydrofolate rcduaase (DHFRi and neomycin resistancr f.%uR J ~mding x- quençrtlr. both under the m n m l of an SVUI promoter.

Transfcctions. The mils were t m s f m e d by clccuopontioo (15) and then culnirrd in RPMI lbu3 containing 104 FCS. which was supplementcd €rom day 1 onward with LOO p s m l GA18 (GIBCO-BRL 8urlington. Ontario. Crinach). Rsisfant c lona wcrc isoiared 12-14 days lacer- Tumor C d Rditemtioa .-y. H-59 cells and transfecrruits wert cul-

ruced in SF-RPMI for 24-h and ihcn disperscd and xeded inio 9b-well plates (Falcon, Lincoln Park 'iJI î t 3 dcnsity of T x ld cclkwell and incubated for 51 h wtrh medium containing 5 5 FCS. IGF-1. or heparqte-conditioncd medium prepmd ;S u.e d k k d pr-iousiy ( LI). The cells wcrr p u k d with 0. I mCi ml of ['Hlthymidine (Du Pont C u a k Mkiiciaugri. Ontririu, C~iada) for 18 h. and thymidine incorpontion was monirored as deeiled elxwhcre (If).

Sortbern Blot A d p i s . Cellular RWA was exmatd from H-59 and tnnsfectcd crlk by the phenol~hloroform method of Chomaymki and Sacchi (161 as we descriid in r f c ~ i l eixwncrr t II). Blots werr prob~d uirh 4

ndioLklcd 0.7-bIobse ELYURI rcszriaion fragment of the human IGF-1 reccptor cDS.4 (.berican Type Culture Colleaion Rockvillc MD). mal>red by laser densitometry. and normdized relativc to the 18s rRVA Ligand Binding Assay. TO quanuntc the binding of IGF-1 to the turnor

celIs. uie method of Phillips cr al. ( 1 9 ws i s d as we d c - m h d in demil previously [ I l ) . execpt that cells wcre grown in RPMI containine 5 5 FCS prior to assay.

MetastPsis h y s , Turnor celis werc inoculatcd by the intmplenic route to gineratc expcrimental Iiver metastases (18) or by tail vein injedons to gencratc lung memwws. Liver rnctastaxs wcrr: enurneratcd immcdiaicly aftcr the rcmoval of or-. Lune werc furcd in Bouin's fimative. and nodules were cnumented as rcponcd previously ( 19). For spontsneous rncmsiyis assays. the tumor cells were injectcd 5.c. into the IatcroaWorninal region. The mean turnor diamctcr for individual turnoa was calculateci h m mtjsurtments in two plana at right mgla. Animals were sacrifice& and mewsrjxs were enumcr- ated when the mean mmor diamctcr for the group rlrached 1.1 cm. In some animais. mmor immunogtnici:y u3s 3ssesscd Sy simul~ntous :njec:ion oi l&

ROLE OF IGF-IR IN CARCWOMA m A S f A S l S

wild-type and trarufeacd cclls at symmetrid conttaiatd sites in ibe la- abdominal region.

To study the rote of IGF-1 R in H-59 growth and metastasis in vivo. the tumor cells were transfected with a vector in which LGF-IR cDNA is constitutively expresscd in the antiscnse or (as control) sense orientations relative to the SV40 promoicr. Several neomycin-mis- tant clones were obtained, and two wcrc srlected for funhcr study. WC found thrit the expression of IGF-IR m W A in thc antisense-trans- fecred clone SA-9 was reduccd by 75% relative to parental H-59 ails or the sense-transfeaed donc SS-2 (Fig. 1). This was rcflccted in rcductions of 65 and 754. respeaively. in the number of IGF-1 binding sites on anfisense-transfectcd SA-9 cells (22137 sites/ccll) relative to sense transfected (64.490 sitcs/cell) or parental H-59 cclb (87.297 sitrs,cell). as assessed by a ndioIabeled ligand binding =y. In vitro. ceIl growth in senim-supplcmcntcd (5%) medium was not affected. 3s shown in Fig. 2. This was also confirmcd in a second m a y where 7-(1,5-dimcthylthi~I-2-yt)-tS-diphenyt-tetrau,Iium hromide (10) was used ro monitor ceil -mowth daily for 5 days (results not shown). However. the amisense-transfected cells lost their ability to respond to IGF-1 or hepatocyteconditioned medium, which are highly mitogenic for parental H-59 and sense-transfectcd cells (fig- 2; Ref. 1 1 ).

To test the effect of IGF-IR suppression un the ability of the tumor cells to colonize secondary organs, they were inoculaicd into synge- neic mice by threr different routes. Following inoculation by the intrrisplrnic route. as means of delivering the ccl!~ into the hepatic circulation via the portal vein (18). ai1 the animals which werr injected

Paptidm Concentration (nu)

8

fi5 2. Lw of rcsporiu ro IGF-1 mi hcparocytccoadirioncd rnedzum t HCMi in

IGF-XR snUxnw asnsfmcd celis. .i. rrum-uuved H FY (3. SS-2 (8). a d %-Y t )

ccUs uerc xtdcd in % w l I plates es u akxntntion OC' i a 10' a l l s u c l l mui~ incubateri wiih difftrcnt con~coaatiom ot' IGF-1 ar 5-C tor 72 h. f'HjThyuudinc u z added drnng the lm IS h of incubaiion B. nimor H-59 m d the tr~nsfcacd cclls wcrc in~ubaicd in the absena (m or pmcucc iC) of 5% FCS or wilh HCW (m. and Ihymidinc uptakc m e s u d T2 h Lter. Rciults arc of 3 qmcntativc cxpcrirncnt dune ia niplkatm. FOL ~rlls culturcd in ururnconnining m d u m thc relative inrxc;iws m ('Hith>midhc incorpontron ucn: C O - . :L6-. md !W-iold +ad for dis r~inired with HCM. iks: .%Zr=

2.1-. 4 . 5 . ~ n d 1.2-iuld for H-9. 55-2 md 5.4-b ~ r l l s . rcsp«xi\cI' Bur. SD.

with 3 X id H-59 or sense-tnnsfectcd ceils developed multiple hepatic nodules by day 14. ranging from 81-30 and from 31-26 nodules F r liver. respectively. In conuast. none of the animdc inoc- ulated with IGF-IR antisew-naasfccted cells dcveloped any noduIes for up to 30 days foIlowiog inoculation (Table 1).

in a second eirpcnmental metastasis =y, animals werc inoculated i,v. via the tail vein with 3 X 1@ turnor cells. Prcviously, we have , shown chat H-59 a l l s preferentially colonki: the l u n e when inocu- lated by this route (9). In the prcsent cxpcriment. anirnals injected with parental or sense-uansfccted celis developed multiple lung col- onies withii 21 days of the injection. rangirig from LW-317 and 8 1-357 colonies per lung. respeaively. However. none of the anirnrils

inoculated with antisenw-transfected cells had detectable lung nod- ules (TabIe 1). To test rhe effect of IGF-IR suppression on spontaneous metastasis

h m a prima? site. animals were injetted s.c. with IO0 tumor cells- Anim3ls in al1 groups developed local tumors. although in animals injeaed with IGF-I R antisense-transfected cells. tumor appeannce was delayed (Fig. 3). The animais were sacrificrd when the mean diameter of their local tumors reached approxirnately 1.1 c m (11.1 days for control mice and JO days for mice injected with cintisense crans- fecrantsh Mile SO? i S of IO) of mimais bexinz tumors of parental or xnx-tnnsfected H-59 celis had liver metastases. none of the animals bearing tumors of ancisense-uansfected cells had detectable livzr metastas& (Table 1). The absence of micrometastases was contimçd by histology using iiver paraffin sections (data not shown). Nonhern biot anaIysis of cells derived from th- tumors when the anirnals were sacrificed confumed thar IGF-IR mRNX ievels in the tumors remained suppressed (Fig. Lb).

In a second experimcnt, snimds were injected s.c. with 2 :K i(I tumor crlls. In rhis sxperirnent, al1 anirnals injected with tumor cells deceioped tumors which grcw rapidly and metastsized to the tivcr in 1005 of the mice. However. the animals injected with anusense- tmnsfrctsd cells did not dcveIop nimors for up to 70 days follow.ing inoculation (Table 1). suggesting tbat 1GF-IR wcis also involved in regularing H-59 growth in the subcutis.

Similar findings were recently reported by Resnicoff er al. (7) using the C6 rat glioblastoma. In these and other srudies with the glioblas- roma model. the reduced tumorigenicity of cells uansfecred with IGF-1 or IGF-1R amisense cDNA was amibuted to increased immu-

jgeniciry of the tnnsfectrd ctlls (6). To trsr xhether the host immune response also played a rolc in the failure of SA-9 cells to develop local tumors when injectcd with the lower dose of 2 X 10' crlls. Ge rechallenged the rumir-free mice with a second inmlurn of I~ wild-type H-59 crlls 75 days after the first injection. Additionally.

WC injectecl H-59 and antiscnsc-transfcaed œlls simultuiwusly at -ai. contraiaicd sites of ihc Iateroabdomiiul region- In both expcrimcnts, parental H-59 cells formcd local nimors. which werc indistinguishable in t h e of q p u a n œ and rate of growth h m tumors in the concsponding control gioups. suggcsting that the mti- sense-uansfcctcd çclls did not indue an effective immune rcsponse in these animais (Table 1).

Our results show tbat H-59 ceils expressing IGF-1R a n k n s e RNA lost iheir ability to metastasüc spontaneously to ~ h c liver or lung nom p r i r n q s.c. tumors and could not colonüe these organs. cven when inmlarcd ditectly into their rnicrovasculature. These results are in agrctmcnt with several reccnt snidies which implicated the IGF-lR in the conml of tumor growth (5.7) and funher extend thtse finding by showing that KiF-IR can play a crucial role in the regdation of mmor ceII potcntial to disseminate and form metasrases in secondary organs.

!Several rnechanisms may be invoked in interpreting our findings. Previously we reportrd that H-59 celis do not p d u c t detrctable tevels of IGF-1 but express high levels of IGF-1R (Ref. 11: Fig. I L The cells require. therefore. an çxogenous source of [GF-1 for acti- vation of the IGF-1R signal transduction pathway. This dependence on pafacrine mcchanisms rendes the livcr and lune favorable target o r g a ~ for rnetastasizing H-59 celfs because hi& levels of IGF-1 are producrd in both organs ( 2 1 )- In celis expressing IGF- L R antisense RNA, the growth advantage in thest sites is apparently lost This may lead to tumor cell death dur: to apoptosis. a proces of ce11 death which c-n nonnally be inhibited by [GF-Li? (22). or it rnay increase nimor cell vulacrability to natural host rcsistance rnechanisms. including the nunoricida1 effecu of resident Kupffer ceils in the Iiver and alveolar macrophages in the lung (19). with a rcsultinp rapid clearance of the cells from the local circulation. tt remains to bç determined. howevsr. whether the failure 01 IGF-LR-suppressed celIs :O rnetastasize is >et another manifecation of the critical role this recrptor and its ligand play in _erow~h conuol or whether other tumor ce11 propenies essential for m e t ~ ~ s formation. such as invasiveness. are also affected. The

O 10 20 30 40 50 Days

fi& 3. Grovrth of H-59 and pinsfmed cclls in wtvo. One million H-59 (C). SS-2 (.), and SA-9 (CI alk wcrc injrctcd +c inm CSïûL6 mier The m u n turnor Climcrcr for indmdiul nimors iwas calaahltd from mriwûcmcnts in NO p h n a st right angler To dctcminc m a n NmOr dkrnctcr (kn. 5 0 ) for die -mup. the sum of fhc individuai mcasurcrnenrs ws diwidcd by the number of iumorhemng ( 11) of 10) m i e

following mu l t s argue in support of tbc latrcr: (a) the p w t h of antisense tnnsfected cells in vitro in serurn-supplemented medium was unchanged relative to conuols; and (b) the amisense tnnsfectants which grew s.c. and gave rise to prïmary tumors failed to rnetastasize to the Iiver or lung (Table 1; Fig, 3).

The failure of IGF-IR antknse-transfected cells to givc rise to tocal tumors folIowing the S.C. injection of 2 X KIS but not 10b celis s u s e s t s that IGF-1 R is atso a Iimiting factor for mmor cell survival and g r ~ w t h in the subcutis and that. in rhis microenvironment, the

- requirement for IGF-1R can bt overcorne undcr conditions of high cell density. as has also k e n suggested prcviously (2). ft is possible th& at high cell densicies, ceII growth in the subcutis could pmcred through autocrine growth mechanisms or other paracrine factors in- dependent of IGF-I (3 3). Alternatively. since the antisense-tram- fectrd cells expressed residual low lrvels of IGF- 1 R (Fig. 1 ). it is alsu possibie that. under conditions of high cell densip. this was sufficient tu r c x u r a srnall subpopulation of the ceIIs expressing the required ibresho1d levef of IGF- 1 R: these nlis cvcntually e i n g rise to the s-c- tumors. Tfiis is consistent with Our observarion thar. in animals in- jected wirh IO" antisense-tnnsfected cells. the latent pend preceding the spperirmce of tumors was considenbly prolonged as compared to conuols. It is also in accordance witb tbe rcported role of IGF-1R in the control of cell survival through inhibition of apoptosis (22)-

ln r-icu of these t'ïnding. the possibilily a ~ o t be rukd out chat the cornplete failure of IGF- i R antisense-UaaSfecfed ceils IO colooize the Iiver or lung is elated to the conditions inhewnt in the rnrrasntic procrss, i.c. the rirrest in the microvascular k d of s d numkrs of single ce& andor s m d m o r ceil emboti (23). This impties that niicrometas~â~s- are Iikcly to be highly susceptible to targeted disrupaon of _mwh procrsxs during these early stages of diimination.

S r w approaches for m c e r therapy are k i n g dcveloped that aim to intenene rit various ';teps in growth factor rrceptor-signaIing prith- ways. One of the most prornisinp targets of more specific thenpy is the r o w t h t'ircror receptor itself. the activit) of which a n br sup- pressed by monoclonal antibodies inducing recrptor down rcgulation (Y) . b' rrceptor kinase inhibitors (14). or by the use of an snt isenx stntesz_v (6 . 7). Our data suggest that. for tumors which are dependent on IGF- 1 for proliferation. the IGF-I R could providr a specific t a r p tor rffscrivs antirnetastatic thcrapy.

References

Li Long. Replotion O/ TUIIKK CeU Ibasion and .WlorlruU by tk T j I Idin-iiAr G r o d Fator RLcepror (IGF-f R)

Chapter V

The Role of Insulin-like Growth Factor I Receptor System in Cancer Metastasis

(11)

Enhanced Invasion And Liver-colonùstion In

Lung Carcinoma Cells Overexpressing the

Insuiin-like Growth Factor 1 Receptor

5.1 m i e w

The role of the IGF-IR in cancer metastasis was M e r studied by transfecting M-27

ceiis with a plasmid vector expressing full length human IGF-IR cDNA. The

invasive/metastatic properties of the transfectants were then analyzed. The d t s of these

anaiyses are presented in this chapter in the form of a submitted manuscript. 1 was

responsible for ai l of the experimentai work described in this chapter with the exception

of the construction of the plasmid vector expressing HGF-IR cDNA, which was

performed by Dr. Raphael Rubin and coiieagues at the Jefferson Medical Coiiege,

Thomas Jefferson University in PhiIadelphia

We found that the transfêcbnts had an enhanced proliferative response to IGF-1 and

hepatocyte-conditioned medium and an i n d clonogenic potential in semisolid

medium. Moreover, they acquired an invasive potential as demonstrated in a reconstituted

basement membrane (Matrigel) invasion assay. When inoculated via the splenic/portai

route in vivo, these celis gave rise to multiple tumor nodules which were not seen

following injection of cells transfected with the vector alone.

Li Long. Reguiation of Tmor Cell f m i o n attd.Crasrasis by r / w Typt I InmIin-iike Growfh Factor Renpror (IGF-IR)

The receptor for the type 1 insulin-like growth factor (IGF-IR) and its ligands IGF-1 and

IGF-iI play important roles in the maintenance of the malignant phenotype. In previous

studies with two sublines of the Lewis lung carcinoma (H-59 and M-27, expressing high

and low levels of IGF-IR, respectively) we have shown that receptor expression in these

tumors correlates with the metastasis to the liver. When IGF-IR expression in highiy

metastatic H-59 celis was suppressed by antisense RNA, the metasbtic potentid was

abolished. In the present study we asked whether the metastatic pmperties can be

modulated by overexpression of the receptor. M-27 carcinoma ceils were transfected

with a plasmid vector expressing fidi length cDNA for human IGF-IR High expression

9 of human IGF-IR in the stable transfectants was confumed by RT-PCR,

immunoprecipitation analysis and a ligand binding -y. These cells had an enhanced

proiiferative response to IGF-1 and hepatocyteanditioned medium and an i n c r e d

clonogenic potential in semisolid medium. Moreover, they aquired an invasive potential

as demonstrated in a teconstituted basement membrane (Matrigel) invasion assay.

When inoculatecl via the splenidporîal route in vivo, these cells gave nse to multiple

tumor nodules which were not seen foilowing injection of cells transfected with the

vector alone. The results suggest that IGF-IR c m modulate several cellular fiuictions

which impact on the metastatic potential including invasion and liver-colonization.

Li bng. Regularion of Twnor Cell Invasion anâ Metarash by the TLgc I I~uIin-likc Grouth Factor Recepror (IGF-IR)

Introduction

Invasion and metastasis are the most life-threatening aspects of malignant disease. The

process of metastasis involves multiple sequential interactions between the disseminating

tumor cells and host tissue. These include cell-ce11 and cell-extracellular matrix (ECM)

interactions mediated through specific adhesion reccptors such as integrins (393),

degradation of ECM by enzymes such as metallo- and serine proteinases (386) and the

regulation of tumor ce11 proliferation by host cell-denved growth factors (1 1).

The receptor for type 1 insulin-like growth factor (IGF-1R) and its ligand IGF- 1 play a

crucial role in the regulation of cellular proliferation, apoptosis and transformation (59).

Recent studies using antibodies to the receptor (347), IGF-1 analogues (345) as well as

antisense expression vecton to IGF-I (394) or IGF-IR (346) have provided evidence

that the IGF-I receptor is involved in the induction and maintenance of the transfonned

phenotype. Using an IGF-IR antisense expression vector, we have previously shown that

suppression of IGF-lR expression in the highly metastatic carcinoma H-59 cells

abrogated their metastatic ability (395). Simila. results have recently b e n reported by

Burfeind et al using a rat prostate carcinoma mode1 (473).

*--

M-27 carcinoma cells are moderately and selectively metastatic to the Iung (387), express

low endogenous IGF-IR and do not proliferate in response to TGF-I(396). We used them

in the present study to invcstigate whether the expression of IGF-IR cauld modulate

metastatic potency, particularly the ability of the tumor cells ta colonize the liver. We

found that ovetexpression of the receptor augmentai both invasion and liver colonization

by îhese ceiis.

Li Long. ReguIution of Tumor Ceil lkasionand ,WemwU ëy the Typc l f i I in- l ike Growth Factor Rccepror (IGF-IR)

Materials and Methods

Cell lines. The origin, metastatic phenotype and methods of transplantation for M-27

cells were describeci in detail previously (387). Briefly, the tumor line was maintained &

vivo by S.C. implantation of lung metastases dexived h m tumor-bearing mice into new

recipient animais. Monolayer cultures were prepared from dispersed tumors and

maintained v i t r ~ for up to 4 weeks (388). Human breast carcinoma MCF-7 celis (397)

were a kind gift h m Dr. M. Pollak (Lady Davis Research Institute, Montreal, Canada).

Ail cultures were maintaineci in RPMI medium containhg 10% FCS (RPMLFCS).

IGF-1R plasmid. The pCVN piasmid vector expresshg a fidl length human IGF-IR

cDNA under the control of the SV-40 promoter was a kind gift h m Dr. EL Baserga

(Jefferson cancer Institute, Philadelphia , PA). The neomycin-resistance gene is also

expressed in this plasmid under the control of the SV40 promoter (269).

Transfection. Transfdon was carried out using Lipofectin, as desmibed elsewhere

(398). Ceils were cultured in RPMI-FCS supplemented h m day 2 onward with

200pg/ml G418 (GIBCO-BR. Canada, Burlington, Ontario, Canada). Nwmycin-

resistant clones were isolated 12-14 days later. Control clones were obtained following

transfection of M-27 cells with the CVN vector alone (mock-transfectants).

Tumor ceU prolireration assay. M-27 celis aad transfectants were senam-starved for 24

hr, dispersed and seeded into 96-weii plates (Falcon@, Becton D e c b n , Lincoln Park,

Li Long. Ruguhation o/Tumor Ceil I l l ~ ~ i o n and lHctastzasu by f i te Type i ImIin-like Growrh Factor Receptor (IGF--IR)

NJ) at a density of 2 x 10' cells/well and incubated for 68 h with medium containing IGF-

1 (Upstate Biotechnology Inc., Lake Placid, NY) or hepatocyte-condiùoned medium

(HCM), prepared as we prewiously describeci (396). Ce11 proliferation was measured by

the MîT [3- (4,s-dimethylthiazol 2-yl)-2,5aiphenyltetrau,lium bromide] assay as was

RT-PCR The reverse transcription &on was performed with 2pg of total cellular

RNA extractecl as we prevïously describeci (396). For amplification, primers

correspondhg to genomic sequences specific for the human IGF-1R (with no homology

to rat IGF-1 R) (269) were used; a sense primer spanning nucieotides 3334-3359 and an

antisense primer comsponding to nucleotides 41 39-4160. Amplification was carried out

in a PTC-100 Programmable Thermal Controiler (Atomic Energy of Canada Ltd.

Montreal, Que., Canada) using estabfished procedures (400). Thirty five cycles of

amplification were pecfonned consisting of denaturation at 94OC for 1 min, reamealing at

55°C for 1 min and extension at 72°C for 1 min, followed by incubation at 72OC for an

additional 5 min. The amplifieci DNA firagments were anaiyzed on 1.2% agarose gels.

The PCR-amplifleci 826-bp product was purifieci and cloned into the pMOSBlue vector

(Arnersham Canada Ltd., Oakville, Ont., Canada) for sequencing. Sequence analysis

was performed by the McGill Sheldon Biotechnology Center (Montreal, Que., Canada)

using the Thenno Sequenase fluorescent labeled primer cycle sequencing kit (Amersham

Canada Ltd., Oakville, Ont., C d ) . Primers corresponding to sequences in the muMe

p-actin gene were used as intemal controls (401). Total RNA fiom the human breast

carcinoma ce11 line MCF-7 was used as a positive control for MGF-IR expression (397).

Ligand binding issay. To quantitate IGF-1 binding to the amior cells, the method of

Phillips et al (402) was used as we previously describeci (395,396) except that celis were

cultured in RPMI medium containhg 5% FCS prior to the assay. Data analysis was

performed using the Ligand program (43) .

125 Immunoprecipitation of IGF-IR Tumor cc11 d a c e proteins were 1-labeled uskg

the lacto-peroxidase method (404). Equal numbers of cells (10') h m each ceil line were

lysed for 15 min on ice with 0.5% NP40 containing ImM PMSF, lmg/ml leupeptin, and

lmg/rnl pepstatin A (ail h m Sigma). The lysaies were cleared by centrifugation at

10,000 g for 15 min. Immmoprecipitation was d e d out using the murine anti-human

IGF-1 receptor MAb aiR3 (Oncogene Science inc., Manhasset, NY) and a rabbit anti-

mouse IgG-saturateci protein A coupled to CL4B Sepharose beads (Phamuiçia Biotech).

The beads were washed repeaîediy with 0.1% NP 40 in PBS, resuspended in SDS sample

b a e r containhg 100 mM dethiothreitol (ICN Biochemical Inc., Cleveland, OH), and

boiled for 15 min. The eluted proteins were separated on an 8% SDS-polyacrylamide gel

and the gels dned and exposed to X-ray film (X-Omat AR, Eastman Kodak Co.,

Rochester, NY) for 7-14 days.

Li Long. Reguhtion of Tmor Ce11 fmasion and Metasrasu by the Type / fmfin-like Growh Factor Receptor (IGF- f R)

a Anchorage-independent growth assay. To mesure anchorage-independent growth, a

modification of the standard soft agar cloning assay was used (405). Briefly, tumor cells

were mixed with a solution of 0.8% agar (Difco Labotatories Inc., Detroit, MI) added to

an equal volume of a 2x concentrateci RPMI-FCS medium, and plated in six-well plates

(Fisher Scientifïc Canada, Montreai, Que., Canada) precoated with 2% agar at a

concentration of 103 ceiidweil. The overlay was aliowed to solidify and then

supplemented with lml RPMI-FCS. The medium was replenished on aiternate days for

12 days. Colonies which exceeded 250 pm in diameter were enumerated using a

microscope equipped with an ocular grid (Diaphot-TMD Inverteci, Nikon Canada).

CeN invasion assay. Turnor celi invasion was assessed in v i t r ~ by the recortstituted

basement membrane (Matrigel) invasion assay as we previously described (392). Bnefly,

Matrigel (Collaborative Researchh, Bedford, MA) diluted to a concentration of 0.23 mglml

was applied to 8 p filters. To each filter 5x10~ celis in semm fke (SF) -RPMI

containing 0.2% BSA were added, Rat fibronectin (5 pg/ml, Gibco BRL) was used as a

chemoattraçtant in the lower chamber. Incubation was for 48 h at 37OC. After removal of

the cells fkom the upper d a c e of the nIter with a cotton swab, the cells on the lower

surface of the filter were stained with 0.2% crystal violet and counted with the aid of a

Nikon inverted microscope. Duplicate filters were used for each ce11 type and 20 random

fields were counted per filter. To control for changes in ceil migration, some fiiters were

coated with 7.5 pg/fïlter of human placenta] type IV collagen (Sigma) which does not

constitute an invasion barrier (392).

Liver colonization way. Mice were anesthetized by an i.p. injection of 28.6 mgkg

sodium pentobarbital. Tumor ceiis (103 w m inocdated by the intrasplenic@ortai mute

to generate experimental tiver metastases (406) and the animals splenectomized 1 min

Iater. Mice were sacrificed and the livers exadneci 16 days later. The metastases were

enumerated immediaîely, without prior fixation.

Statistics. The Stuâent t-test was used to anaiyze ciiffierences in cloning efnciency and the

ManuWhitney test to analyze metastasis data.

M-27 cells were transfected with a plasmid vector expressing a full length cDNA for

human IGF- 1 R under the control of the SV 40 promoter. Control cells were transfected

with the vector alone (mock-transfectants). Neomycin-tesistant clones were obtained and

several were selected for £ûrther study. Expression of human IGF-1R mRNA in these

clones was confirmeci by RT- PCR using primers specifk for the human IGF-1R

sequence (269) for ampiificaîion. An 826-bp PCR product couId be amplified h m

RNA obtained b m clones R3, R4, R9 and R19 and h m the human carcinoma MCF-7

cells (a positive contml) but not h m wiid-type and vector transfected M-27 cells, or

fiom 8 5 9 celis, although the latter express high levels of the endogenous moue IGF-IR

mRNA (Fig la) (395,396). DNA sequence anaiysis confhmed that the PCR product was

amplined h m human IGF-IR RNA. In addition, MA\, aIR3 to human IGF-IR

immunoprecipitated two bands of Mk 112,000 and 94,000 corresponding to the

molecular masses of the a and B subunits of human IGF-iR, h m ceii lysates of R3 and

R4 but not h m mock-transfected and wild-type M-27 cells or h m H-59 cells (Fig lb).

Finally, a ligand-binding assay using '%IGF-1 demonstratecl that the transfection

increased the IGF-1 binding sites h m 21,214 sites/ceii in M-27 cells to 67,487 sitedcell

in R3 cells.

The effect of increased IGF-IR expression on several cellular fllnctions which could

impact on the metastatic potentiai of the cells aamely, cell pmlûeraton, clonogenicity

and invasion was then assessed. We found that the IGF-IR aransfectants acquired a

Li Long. Regdation of Tiuniw CeII I-ion a d ,Uèm&uu by rlY T j p I ImI in- i ik Growth Fpcror Recepror (IGF-IR)

0 mitogenic response to IGF-1 and to hepatocyte-conditioaed medium (Fig 2), two

properties also characteristic of the liver - metastasking H-59 cells (395, 396). In

addition these ceii had an increased cloning efficiency in semisolid as compared to M-27

(3-4 fold increase) or mock transfected (7-8 fold) cells (Table 1).

When the invasive potential of the cells was assessed using nucleopore filters coated

with recomtituted basement membrane (Matngel), we found that the HGF-IR-

transfectants were 8-10 fold more invasive than e i t k Ad-fype or mock-transfected M-

27 cells (Fig 3).

Finaliy, the effect of IGF-1R overexpression on the ability of M-27 cells to colonize the

liver was evaluated 16 days foliowing tumor ceil injection via the intrasplenidportal

route (406). None of the animais (0/5) inoculateci with mock-transfected CVNl and

CVN3 celis developed any liver nodules while al l the mice injected with clones R3, R4

and R9 celis developed multiple hepatic tumor noduies (median numbers ranging h m

7 1-1 10 nodules per liver, see Table 2). Only 10/13 animals injected with wild-type M-27

cells developed hepatic metastases and the median numbers of metastases in these mice

was 5 (jH0.01).

Li Long. Regufation of Tumor C d Imtasion d ,Wtastasis by the Tjpe /frrnrf~-likr Growrh Factor Receptor ([GF-IR)

Discussion

The IGF-IR has been strongly implicated in the induction and maintenance of the

transformed phenotype (see review in ref. 59). Ovetexpression of this receptor in

different ce11 types lead to induction of a t d o r m e d phenotype (327, 328), while the

loss of its expression was shown to renders cells resistant to transformation and could

reverse the transformed phenotype (325). However the d e of this receptor plays in

regdation of the invasivefmetastatic phenotype is still poorly understood (407).

Previously, wc reported that suppression of IGF-IR expression in highly metastatic H-59

ceiis c a w d an abrogation of the metastatic potentiai (395). We demonstrate here that

overexpression of KGF-IR in turnorigenic cells can enhance their invasiveness and

increase their iiver-colonizing potentiai. Thus the d t s extend our previous fïndings and

implicate the IGF-1 receptor in several ceiiular ftnctions which can impact on the

metastatic potential including cellular prolifition, anchorage-independent growth,

invasion and the ability to grow in the hepatic microenvin,nment

Severai mechanisms may be invoked in inîerpreting our fïndings. The increased liver-

colonking potential of the transfectants may be directly relateci to the increased

proliferative response to IGF-1. This may provide the cells with a growth-advantage in the

hepatic microenviroment and therefore with the ability to more effectively evade

resident host defense mechanisms (e.g., tumonciclal Kupffer celis). On the other hand the

increased invasiveness of the cells may facilitate theù extravasation into the parenchyma

positioning them in closer proximity to the IGF-1-producing hepatocytes (20). IGF-1 has

Li Long. UeguIatlion of Tumor CelI Invarion and ,Wemmasis by rliv Typr I wuf fin-like Growth F ' o r &cepror (IGF-I R)

O also been identifïed as ce11 swivai factor (408) and the metastatic phenotype has been

linked to increased resistance to apoptosis (336). It is conceivable therefore that IGF-1-

mediated suppression of apoptosis, which in turn may be induced by local cytokines (e.g.,

Kupffer ce11 derived TNFa) provides tumor cells overexpressing IGF-IR with a suMval

advantage in the liver.

Unlike other models used to investigate the role of IGF-IR in tumongenesis (e.g.,

fibroblasts), M-27 cells are tumorigenic and moderately metastatic to the lung.

Interestingly, in these ceUs overexpression of IGF-IR did not result in a signifïcant

change in either tumongenicity, m e d as the incidence and growth rate of

subcutaneous tumors, or the potential to colonize the lung following i.v. inoculation (&ta

not shown). In fact the major effect which IGF-IR overexpression had on these cells

vivo was a marked increase in their liver-colonizing potential to levels normaUy observed

with carcinoma H-59 (395, 396). Recently we found that M-27 cells produce EGF,

PDGFa and PDGFP and express the respective receptors, whereas H-59 cells do not

express detectable levels of EGF or EGFR and have signincantly lower levels of PDGF

and PDGFR (please see chapter IV, Fig 4-1). growth of M-27 cells v i v ~ may therefore

be regulated in an autocrine fashion by PDGF and EGF. IGF-1 is a progression factor

which acts in concert with cornpetence factors such as PDGF to drive the ce11 into S

phase (1 1). Since M-27 celis do not produce detectable levels of IGF-1 (chapter IV, Fig

4-la), the celis which overexpress IGF-IR depend on an exogenous source of IGF-1 to

activate the IGF-IR signaling pathway. It is conceivable therefore that the growth-

Li Long. Regcbtion of T w r Celt Invusion and iU~14stas~ by the T m I /miin-lik Grorvth Factor Receptor (iGF- I R )

a promoting effect of IGF- 1 R on these celis could best be manifest in a microenvironment

with high levels of IGF-1, such as the liver (20). Thus, regionai (Le., organ-specific)

differences in the relative abundance of various growth factors, as weil as the repertoire

of growth factors and receptors expressed on the malignant cells may ultimately

determine the potential of disseminating cancer ceiis to co1on.k different organ sites.

Mutationai analyses have recently suggested that the mitogenic and transforming

activities of the IGF-IR are nguiated through activation of distinct signahg pathways

(409). In our system, the overexpresion of IGF-IR redted not o d y in enhanced

mitogenic response to IGF-l a d increased clonogenicity (a measure of the transfomieci

phenotype) but also in incrrased invasiveness. Further studies are required to determine

whether increased c d invasion in this system is the b c t i o d outcome of a distinct,

IGF-1-induced signaling d e .

Tumor invasion and metastasis represent the outcome of a complex series of sequential,

interlinked steps. Recent &ta have indicated that through its binding proteins, IGF-1 can

also modulate ce11 adhesion and consequentiy motility (410). Changes in any one of

these cellular parameters, independentiy of growth, is likely to impact significantiy on

the metastatic potential of the turnor ceiis. The present tumor system provides therefore a

model for M e r dissection of the d e of IGF-IR in metastasis in general, and in

particular its contribution to organ-selective pattems of turnor dissemination.

a Acknowldgmcnts

We are grateful to Dr. Renato Baserga (Thomas Jefferson University, PPhiladelphia, PA)

for helpful discussions and to Mrs. Irene S i d o d o for help in the preparation of the

O ! m w a w m w 1 w m m w 4 O 2 4 6 8 10 12 14

1GF-1 Concentration [nM]

Fig 1. IGF- IR' M-27 ceUs proLiferate in respanse to IGF-1 and HCU a: Sem-starved M-27 (*), CVN3 O, R3 (A) and R4 (O) celis were seeded in 96-weli plues a a concentration of 2 x lo3 celidweii and incubateci with different concentrations of IGF-1 at 37°C for 72 h. MTT was ddd during the last 4 h of incubation- b: CeUs were cultureci in HCM . R e d t s are of a representative experiment curied out in tripkatests Light micrographs d e p i h g colonies characteristidy developed by the twnor celis are shown in c (Next page): 1-4: M-27, CW3, R3 and R4 cuitured in SF-RPMI supplamiaed with SOngIml IGF-1, 5-8: the same ceUs cultureci in HCM.

Fig 2. RT-PCR based analysis of hIGF-IR expression in M-27 transféctants. a: Total

RNA (2pg) isolateci h m M-27 ceiis, (mock-transfected) CVN3 and (receptor

transfected) R3, R4 and R9 ceiis were reverse transcribed and then amplified using hIGF-

IR or B-actin specific primers as detailed in Materials and Methods. RNAs fkom MCF-7

and H-59 ceiis were used as positive and negative controls, respectively. b: Protein

expression was anaiyzed by imrnuuoprecipitatiioe '25~-radiolabeled proteins were

precipitated h m NP40 extracts of M-27, H-59, CVN3, R3, R4 and MCF-7 celis using

mAb aIR3. The irnrnunoprecipitated proteins were separated by electmphoresis on 8%

polyacrylamide gels under ducing conditions and the protein bands visualized by

autoradiography. The estimated molecular weights are indicated on the Ieft.

Fig 3. Increased invasiveness in tumor d i s overexpressing IGF-IR Ceils (SX lo4 in 100

pi medium) were dded to Matrigel-coated filters in scnim-hec d u m and iaaibated for

48 h at 37OC. Resuhs are presented as percent of invasion reiative to control ceils. Bars

denote SD.

e Table 1. Increased anchorage-independent growth potential in tumor ceils overexpressing

IGF- IR Cells were cloned in semisoiid agar as descnied in Materials and Methods.

Shown are the means and SD of triplicate plates. The cloning efficiency of R3 and R4 cells

were signifjcantly higher than that of M-27 celis (P<0.0025 and P<0-01 respectively). The

cloning efficiency of CVN3 cells was lower than parental M-27 cens (P<0.025).

Cell Line No. of ColoniesiPlate MeanBD

Table 2. Enhanced tiver-colonizing potential of M-27 ceUs overexpresshg IGF- 1 R

Animds- were inoculated with 10' -or c e b by the intrasplenidportal route and

splenectomized 1 min later. Livers were analyzed 16 days d e r the injection. The results

are expressed as medians and (range). The number of liver metastases in alI animals

injected with hIGF-IR' M-27 celis was signincantly higher than that in rnice injected with

Cell Tumor Cell Metastases NoduleslLiver Line lnoculum (Incidence)

(cellslmouse)

Chapter VI

The Role of Insulin-like Growth Factor I Receptor System in Cancer Metastasis

(W

Regulation of Mr 72,000 Type IV Collagenase Synthesis by the Type 1 Insulin-like Growth

Factor Receptor.

Li Long. Regufarron ~J-Tumor Cell Invasion ami .Wettumsu by r h e Typr I I ' i n - I t k e Grovth Factor Rccepror (IGF-IR)

The findings descnbed in this chapter demonstrate that IGF-1 regulates the expression of

the Mr 72,000 type IV coiiagenase (MMP-2, gelatinase A). They are presented in the

form of a submitted m m d p t . 1 was responsible for dl of the experimental work

descnbed in this mmuScTipt

In this study, the expression of MMP-2 was investigated in tumor H-59 and M-27 celis

and the respective IGF-IR (antisense or sense) transfectants (see chapters 4 and 5). H-

59 celis in whkh IGF-IR expression was suppressed by transfection with an IGF-1R

antisense expressing vector were signincantly less invasive than wild-type or sense

controls in a reconstituted basement membrane (Matrigel) model. These ceiis expresed

lower levels of MMP-2 mRNA and protein as assesseci by RT-PCR, Western blot

analysis and gelatin zymography. Conversely, overexpression of IGF-IR in M-27 ceiis

with low endogenous levels of the receptor caused a marked increase in MMP-2 mRNA

expression with a comspoeding increase in the levels and activity of the protein. In both

models the addition of IGF-1 to the culture medium of receptor-expressing ceiis resulted

in the production of MMP-2 M A .

Li Long. RcgirIation of Timor Ceil Invasion and ,Uerm~pris by th 7' I hfin- i ike Grouch F4ctor Rcccpror (NTF- I R )

CIassz~cafion: CeU Biology

Reguiation of Mr 72,000 Type IV Collagenase Synthesis by the Type 1 Insuiin-like Growth Factor Receptor.

(matrix metaiioproteinases / type N collagenase / type 1 insulin like growth factor / invasion / metastasis)

LI LONG, HUA LING AND PMNA BRODT'

Department of Surgery, Division of SUtgicai Rcsearch, McGiIl University, Royal Victoria Hospital, Montreai, Quebec, Canada H3A 1Al

1. To whom the quest for reprint should be addressed.

Abbreviations: MMP, mat& metallopmteinase-, IGF-1, type 1 insulin-lïke p w t h factor, IGF-1R receptor for type 1 insulin like growth factor, EGF, epidermai growth fàctor; TIMP, tissue inhibitor of metalloproteinase; SFM, serum fk medium.

Address for correspondence:

Dr. P. Brodt, Surgical Research, Royal Victoria Hospitd, 687 Pine Ave. W., Room H6.25, Montreal, Quebec, Canada H3A 1Al

Abstract

We învestigated the role of the type 1 insulin-Wre growth factor in regulating tumor ce11

invasion and the synthesis of the 72 kDa type IV collagenase @Mi?-2). Highly invasive

Lewis lung carcinoma subline H-59 cells in which expression of the receptor for type 1

insulin like gmwth factor was inhibited by antisense RNA had a signincantly reduced

invasive potentiai in a reconstituted basement membrane Matrigel) invasion mode1 as

compared to controls. These cells expresseâ lower Levels of MMP-2 mRNA and protein

as assessed by RT-PCR, Wesfern blot d y s i s and gelaîin zymogmphy. Conversely,

overexpression of IGF-IR in a second, p r l y invasive carcinoma subline, M-27 with low

endogenous levels of the receptor caused a d e d i n c m in MMP-2 mRNA expression

with a correspondhg increase in protein Levels and invasion. Finally, ligand-mediated

activation of IGF-IR signifïcatltiy increased MMP-2 synthesis in both celi types. The

results idenifL IGF-1 as a regulator of MMP-2 expression and cellular invasion.

0 The receptor for type 1 insulin-like growth factor (IGF-1 R) and its ligand play a critical

role in the regdation of cellular proliferation, apoptosis and transformation (59. 328). We

are investigating the role of the receptor in the regdation and maintenance of the

metastatic phenotype using a murine carcinoma mode1 of two sublines of the Lewis lung

carcinoma, H-59 and M-27, with divergent potentials to metastas* to the Liver which

correlate with IGF-IR levels. Previously we reported that H-59 cells expressed higher

levels of the Mr 72,000 type N collagenase (MMP-2) tban M-27 cells and this correlateci

with invasiveness as measured in the reconstituted basement membrane (Mairigel) assay

(392). Suppression of IGF-IR expression in H-59 cells by stable transfection with a

plasmid vector expressing IGF-IR antisense cDNA abrogated their metastatic potential

(395) while overexpression of IGF-IR in M-27 cells d t e d in enhanced invasiveness

and an increaseû potentiai to colonke the liver (Long, L. et al, m. submitted).

The dissolution of extracellular matrices by proteinases is an essentid step in the process

of metastasis. The matrix metailoproteinases (MMPs) are a f d y of zinc-binding

proteinases which play a role in processes such as idammation and wound healing.

Severai memkrs of this farnily, including type 1 collagenase (MMP-l), the gelatinases

(MMP-2 and MMP-9) and stromelysins (MMP-3 and MMP-IO) have been implicated in

cancer ce11 invasion (122, 411). The evidence is particularly compelhg for the

involvement of MMP-2 in the dissolution of basement membrane barriers, a process

thought to be required for tumor ceii invasion into blood vessels and for tumor

extravasation (8 1, 122,412). MMP-2 and otha mdoproteinases may also facilitate the

0 expansion of a growing tumoi m a s in the primary site either directly by proteolytic

cleavage of ECM proteins or indirectiy by activation of other proenzymes (122). These

enzymes can also activate latent, ECM bound growth factors &or inactivate growth

inhibitory molecules (122,370,412) thereby indirectly affecting cellular proliferation.

The fkding that in our mirrine carcinoma model, the invasive and metastatic potentials of

the cells correlateci with IGF-IR expression and MMP-2 b e l s prompted us to investigate

whether the IGF-IR/IGF-1 axk plays a role in the regdation MMP-2 synthesis. To this

end, we used clonal populations of the tumors in which IGF-IR levels were either

suppresseà @-59) or enhanad (M-27) by gene transfer. Our evidence suggests that IGF-

IR can regdate the expression of MMP-2. This implies that in addition to its p w t h

moduiating effocts, IGF-1 can also impact on the metastatic potential by increasing the

collagenolytic activity of the ceiis.

Li Long. Regrrhion of Tmor Ccll f m i m and Muttasu by the T m I Insufin-like Crowth Factor Reccrpror (iGF-I R)

Materiais and Methods

CeII lines. The origin, metastatic phenotypes and methods of transplantation for tumors

H-59 and M-27 were d e d b e d in detail previously (387). Transfection was perfonned

using Lipofectin (398). Culture medium was supplemented with 200pg/ml G418 two

days after the ttansfêction and thereafter. Al1 tissue culture reagents were obtained h m

Gibco BRL (Burlington, Ont, Canada), SA-1, SA-8, SA-9 and SA40 were clonal lines

derived h m H-59 ceils transfected with the CVN plasmid expressing IGF-1R antisense

cDNA; Clone SS-2 was derived h m H-59 ceiis transfited with the same plasmid

expressing the IGF-IR cDNA in the sense orientation. The origin and phenotypes of

these clones were described in detail previously (395). Clones R3, R4 and R9 were

0 denved h m M-27 cells transfected witb îhe CVN vector expressing fbll length human

IGF-lR cDNA; CVNl and CVN3 are control clones derived h m M-27 ceiis transfected

with the vector only. The NM 3T3 mouse fibmblast ce11 line was obtained h m Dr.

Clmord Stauners (McGill Cancer Center, McGill University, Montteal, Canada). It was

maintained in RPMI supplemented with 10% fetal calf~mnn.

Ceil invasion assay. Turnor ce11 invasion was assesseci in v- using the reconstïtuted

basement membrane (Matrigel) invasion assay as we described in detail elsewhere (392).

Matrigel (Collaborative Research, Beaord, MA) was used at a concentration of 0.23

m g h l and rat fibronectin (5 pg/ml, Gibco BRL) was used as a chemoattrstctant Cells on

the lower surface of the fiiter were enumerated using a Nikon inverted microscope and

Li Long. Reguiation of Twnor C d Iiriwion and .%ferastasis by the T7yprr I Insuth-fi& Growth Factor Uece~tor (KF-IR)

duplicate samples were analyzed for each assay condition. To control for changes in ceII

migration some fiIters were coated with 7.5 pg/nlter of human placentai type IV coiiagen

(Sigma-Aldrich Canada, Ltd., Mississauga, Ont., Canada), a concentration which does

not constitute a migration barriet (3).

RT-PCR Total cellular RNA was e-ed using the procedure we previously described

(396). Two pg of totat RNA were reverse transcribed using a cocktail of 50 mM Tris-HCI

(pH 8.3), 30 mM KCl, 8 mM MgC12, 10 m M Dm, 2 mM each dNTP, 50 ng/ml of

random hexadeoynucleotide primer, 8 U / d RNase inhibitor, and 8U of aMan

myeloblastosis virus reverse traasaiptase (aii h m Phamuicia Biotech, Baie D'Urfe,

Que., Canada). The mixture was incubated for 10 min at 23OC, then for 45 min at 42°C

and finaily for 5 min at 9S°C. One-tenth of the cDNA product was used in the PCR

reaction Primers speciflc to mouse MMP-2 were designed h m the lcnown cDNA

sequence (413). The sequence for the upstream primer comsponded to nucleotides 1381-

1401 and the sequence for the dowmtream antisense primer comsponded to nucleotides

2099-2 1 19. Using these primers a 738-bp product was amplifïed which hybridized to a y-

S2p]~TP end-labeled MW-2 oligonucleotide (bp 1742-1769) in a Southern blot assay.

Two primers for the murine actin gene (correspondhg to bp222-240 and 1052-1070)

were used as intemal contmls (401). Thirty-five cycles consisting of 1 min incubation

each at 94OC, 5S°C and 7Z°C were useà and followed by a 5 min incubation at 72OC.

The amplined DNA hgments were analyzed without fiutha purification by

Li Long. Regufatron of Tmor Cell I d o n and Metauasis by r /w T m I Ihuiin--lik Growrh Factor Receptor (rGF-f&

electrophoresis on 1.2% agarose gels. Total RNA h m mouse fibroblast NIH 3T3 cells

was used as a positive control for MMP-2 (41 4).

The optimal number of PCR cycles for which exponentiai production of the PCR

products of interest (MMP-2 and p-actin) can still be observeci was detennined in

preliminary experiments using NIH 3T3 RNA (Fig Sa). Reverse transcribed tumor ce11

RNPL was then amplifieci using 35 cycles to ensure a linear range. Because the quantity of

amplined p-actin hgments is assumed to be proportional to the initial amount of mRNA,

the relative level of expression of MMP-2 wuld bc detennined by normalizing to the p-

actin level, using densitometry.

Western blot assay. To preparr tumot conditioned media, confiuent monolayers (8x 1o6

cells) were washed extensively to remove the senim, the cells culhaal for 72 h at 37OC in

serum fiee (SF) medium with or without 6.67 n M recombinant human IGF-1 (UBI, Lake

Placid, NY) and the supernatants lyophilized and reconstituted (50-fold concentration)

until used. Western blot analysis was c e e d out as we previously described (41 5). The

blots were probed with polyclonal antibodies to MMP-2 and TIMP-2 @th kind gi&

£iom Dr. Stetler-Stevenson, Laboraîoxy of Pathology, NCI, NiH, MD) (17) and the

relative amounts of MMP-2 and TIMP-2 were assessed using an LKB Bromma üitroscan

XL Enhanced Laser Densitometer.

Gelatin zymography. The gelatinolytic activity of MMP-2 was analyzed by ymography

as described elsewhere (3). nie concentrateci conditioned media nom H-59, M-27 and

m. the transfectants were electrophocesed on an SDS poiyacrylamide gel containhg lmg/mi

gelatin. The gels were stained with Coomassie blue and destallied with 10% acetic acid-

50% methano1 until the desired color intensity was obtained. The gelatinolytic activity

seen as a clear zone on the blue background was quantitateci by densitometry-using

photographie negatives of the gels.

Raults

To study the effect of aitered IGF-IR expression on tumor ce11 invasion, the Matrigel

assay was used. The results shown in Fig 1, demonstrate that in H-59 cells the

suppression of IGF-1R expression caused a reduction of up to 50% in tumor ceil

invasion. This was in accord with earlier îïndings that overexpression of IGF-1R in the

poorly invasive M-27 ceils sisnificantly augmented (8-10 fold) their invasive capability

(Long, L. et al, ms. submitted). The major basement membrane degradhg proteinase

implicated in Matrigel invasion is the 72 kDa type IV collagenase (MMP-2) (370,416),

we therefore investigated whether its expression in these cells was altered. Quantitative

RT-PCR analysis was used to analyze changes in MMP-2 mRNA levels. Results shown

in Fig 2(b) indicate that in M-27 celis overexpressing IGF-IR, the expression of MMP-2

mRNA increased 2.5-7.7 fold as wmpand to wild-type or mock-transfected controls.

Conversely in H-59 cells expressing IGF-IR antisense mRNA, MMP-2 mRNA levels

were reduced 2.5-6 fold relative to wild-type or control transfectants. In these ceils the

expression of TIMP-2 mRNA as measured by the Northem blot assay was unaltered (data

not shown).

To assess MMP-2 and TIMP-2 expression at the protein level, tumor-conditioned media

were analyzed by the Western blot assay using antibodies to MMP-2 and TIMP-2.

Results shown in Fig 3(a) confîrmd that in M-27 cells overexpressing IGF-IR, the

increase in MMP-2 mRNA was parailelecl by a comsponding increase (2-4 fold) in

MMP-2 protein levels. Furthexmore. when the cells were serum staived and then cultured

in medium supplemented with IGF-1 (SOngM), MMP-2 levels in al1 cells increased by

2-4 fold relative to unstimulated cells. In the same cells TMP-2 leveIs remained

unchanged (Fig 3b).

In cells overexpressing IGF-IR, the increased production of MMP-2 was refiected in

increased gelatiwlytic activity as assessed by ymography. Results shown in Fig 4

demonstrate that in tumorconditioned media, two zones of lysis corresponding to the

latent (72 kDa) and the activated (66-68 kDa) forms of MMP-2 were present. These

zones of lysis couid not be seai in the presence of EDTA (not shown) indicating that they

were produced by a metallopmteinase. These gelatinolytic activities were more

prominent in medium conditioned by M-27 cells overexpressing IGF-IR than in media

conditioned by wiId-type or mock-transfected cells when the d s were cultiired in the

absence of IGF-1 (10-1 1 fold) and were f.urther increased in dl ceils in the presence of

IGF-1. This stimulatory effwt could not be seen when epidermal p w t h factor was used

(results not shown). Conversely, a reduction (2.5-5 fold) in gelatinolytic activity was

seen in H-59 ceils expressing IGF-IR antisense mRNA as compared to wild-type or

sense-transfected cells. Momvet, while in the latter cells, this activity was enhanced up

to 2-2.4 fold following addition of IGF-1 to the culture medium, no hcrease in

gelatinolytic activity was seen when antisense expressing cells were treated in a similar

manner.

Li Long. Regdarion of Tumor Ce11 l l m i o n and Mctas~pru by the T m CIll~uIin-iike Growrh Factor Recepror IIGF-/RI

Discussion

Proteolytic degradation of the extracellular ma& is an essential and recurring process in

the course of cancer metastasis. Cleavage of type N collagen, the major constituent of

basement membranes permits local invasion at the primary site, is required during tumor

ce11 intravasation and extravasation and is a major trigger for angiogenesis (7). Prominent

among the proteinases involved in bascment membrane degradation are the type N

collagenases or gelahases also known as MMP (matrix metallopmteW )-2 and MMP-

9 (370,386). MMP-2, is secreted as a 72 kDa ymogen and is acdivated extracellularly to

produce partiaüy and M y activated gelatinases of 6268kDa (361). It has been

implicated in invasion and metastasis of diverse tumors (1 22,3 52). Suppression of MMP-

2 activity by synthetic or natural iabibitors has been shown to block -or invasion in

and meîastasis &z vivo (1 22,380,4 17-4 1 9).

The regulation of MMP-2 synthesis in manudian cells is stiii not weU understood.

Unlike other metalloproteinases such as MMP- 1, MMP-3 and MMP-9, MMP-2

synthesis is not inducible by cytokines such as TNFa and I L 1 (367, 420) and is

refiactory to the protein Kinase C (PKC) activator Phorbol-12-myristate-13 acetate

(PMA) (81, 363). Among the factors reported to activate MMP-2 synthesis are the

cytokine TGF-P (81, 363), i n c d concentrations of intracellular calcium (421) and

the ECM proteins laminin -which may fùnction through a phospholipase D activating

pathway (422, 423) and vitronectin which activates signal transduction through the

Li Long. RrguIation o/Tmor CeIf Invasion and ~ l o ~ r a s i r & the T~pr 1 Inntlin-like Growrh Factor ûecepror (IGF-IR)

integrin vitronetin receptor a$3 (362). The role of oncogenes such as Ha-ras and c-erb

in the regulation of MMP-2 synthesis, although it was suggested by some reports is still a

matter of controversy (424,425). One reason for the conflicting reports on the regulation

of MMP-2 may be the involvement of tissue-specific enhancer - promoter elements in its

transcriptional regulation, has been suggested by a ment report (426).

Our r e d t s provide the f h t LUie of evidence implicating IGF-1 in the regdation of MMP-

2

synthesis. They, therefore, add new insight into the d e which the IGF-IR plays in tumor

progression implicating it in the regdation of late events in this process, namely the

acquisition by transfomed cells of an invasive/metastatic phenotype.

IGF-1 has been implicated in transcriptional regulation of various genes including the

early response genes fos (304, 427, 428) and jwi (429). ceil cycle intermediates such as

cyclin-Dl and cdc2 (430) and recently, the urokinase plasminogen activator inhibitor

PAI-1 (43 1). IGF-1 has also been implicated in the regulation of the synthesis of ECM

proteins such as collagen (432) and proteogiycans (433). The findiag that it also regulates

MMP-2 synthesis suggests that IGF-1 could play a dual role in the coordination of ECM

tumover and maintenance of homeostasis. As IGF-1 has been shown to enhance steady

state mRNA Ievels through a direct effect on gene transcription or by pst-transcriptional

modification of mRNA, which stabilize the mesage (431), the precise molecuiar

mechanism involved in regulation of MMP-2 synthesis remains to be elucidated.

The rnolecular events which are triggereci foilowing activation of the IGF-I receptor are

currently the subject of active investigation and are not yet fÙUy understood. The

evidence suggests that multiple signal transduction pathways can be turned on in

response to ligand binding by IGF-IR. They include a tyrosine phosphoryIation cascade

involving IRS- 1 (insului receptor substrate-l), Shc, Grb-2 and mSOS which are linked to

the ras signahg pathway (reviewed in tef. 59), activation of phospholipase C and the

accumulation of IP3. While the link between these signaling intermediates and MMP-2

synthesis remains to be elucidated, it is of interest to note that IRS-1 was recently found

to be associated with the integrin vitronecth receptor a& (434), a modulator of MMP-2

synthesis which was also recently identifIed as a ceii sucface receptor for MMP-2 (435).

The signal transduction pathways activated by IGF-IR and appear therefore to

converge with the likely result of signal amplification and an enhancement of the

proteolybç and invasive capabilities of the c d .

la a recent report, it was shown that the inhibition of TGF-1 P receptor-mediated calcium

influx blocked the activation of MMP-2 transcripption in three different human tumor lines

(421). This suggests that calcium mobWtion is a criticai component of TGF-1P-

mediated activation of MMP-2 synthesis. As ligand binding by IGF-IR is bown to

induce to a rapid accumulation of IP3, resuiting in increased cytoplasmic levels of fke

calcium (436), it may be one mechanisms for IGF-1-mediated regulation of MMP-2.

a The link between IGF-IR and MMP-2 may be reciprocal. Thus, recent studies have

implicated MMP-2 in proteolytic processing of IGF-1 binding proteins 3,.- and 5 resulting

in increased bioavailability of IGF-1 (52, 53, 289) which in may lead to M e r

amplification of MMP-2 synthesis. As IGFBP-S is reportdy incoprated into the

extracellular matrix, it may also provide the celis with an ECM-bound resewoir of IGF-1

(290) which can become accessible during ECM degradation. Enhanced production of

MMP-2 may thereby d t not ody in increased invasion but also in augmented cellular

proliferation.

Taken together with ou. findings, the data suggest that the activities of IGF-1 and MMP-2

are coordinated at the levels of both transcription and fiinction and that together they play

a centrai role in regulat& the metastatic phenotype. This provides a wmpelling

rationale for targeting the IGF-IR and IGF-IR-activated signaling pathways in the design

of anti-metastatic therapy.

Fig 1. The invasiveness of H-59 ceiîs correlates with IGF-IR expression- H-59 ceUs

(5 x 1 O' in 100 pi medium) were added to the Matrigel-coated filters and incubated for 48 h

at 37°C. Resuits are expressed as percent of invasion relative to control cells. Bars denote

SD.

Li Long. ReguIation of Tumor Cell I n v ~ ~ i o n Md Ickmcaru by ht Tjipc I I d i n - l i k e Growrh Facror Reecpror (ICF-IR)

Fig 2. RT-PCR based d y s i s of MMP-2 expression in IGF-IR tdectants . a:

Optimal conditions for quantitative analysis of PCR-based amplification of MM.-2

cDXA were determineci using cDNA derived h m NM 3T3 fibroblast Two sets of

primers for MMP-2 and B-aain and 20-41 amplification cycles w m used. PCR products

were separated by electrophoresis on 1% agarose gel. The yields were analyzed by

densitometry, and the relative yield of MMP-2 proportionally to p-actin was calculated

for each PCR cycle. b: Total mRNA was reverse transcribed and the cDNA amplified

using the same MMP-2 and p-actin primers for 35 PCR cycles. NIH 3T3 fibroblaots were

used as controls to quantitate relative levels of MMP-2. The positions of the size markers

are shown on the 1eR Results of the densitometry are shown were nomializcd relative to

p-actin and are shown in the bottom graph.

Fig 3. Western blot analysis of MMP-2 expression. Media wnditioned by M-27, mock-

transfectant CVN3 and receptor transfected R3 and R4 cells were separated by

electrophoresis on 10% SDS-polyacrylamide gels and the protein transferred onto

nitrocellulose nIteis and pmbed with rabbit anti-mouse MMP-2 (a) and TiMP-2 (b)

antisenr Laser densitometry was performed on transparencies The results of the

densitometric analysis were normalized relative to untreated wild type M-27 ceUs which

were assigned a value 1 and arc shown in the botiom panel. The estimateci molecular

weights are shown on the left.

Li Long. Rrguhtion of Timor CeIl Iùwrion d M e ~ p s m i r by rlic T' 1 I . d & Growrh Factor Receptor (IGF-f R)

Fig 4. Zymographic anaîysis of gelatinssc activity. Conditionad media werr harvested

fkom cells incubated for 72 h with or without IGF-1 and concentrated by 50 fold. Results

of densitometry are shown in the bottom panel. The values were normaiized relative to

the respective unstimulateci, wild type cells. The estimated molecuiar weights are

indicated on the le&

Chapter VI1

Tumors H-59 and M-27 Cells Differ in their

responses to IGF-1 and differentiaiïy Express

other Growth Factors and Receptors-

Summary of Unpublished Results

Li Long. Replatton of Timor Ceif hvasion und .Clor~t&as& by the Type I Insuiin-li& Growth Factor Receptor {IGF-IR)

In Chapter IV, a summary of the phenotypic diffierences between H-59 and M-27 cells

was provided based on published data. Several unpubüshed observations are relevant to

interpretation of the datû described in Chapters IV, V, and VI and are therefore described

in this chapter. 1 was responsible for all of the experimental work described in the

present chapter.

Northern blot analysis was used to investigate growth f&ctor and receptor expression in

the two tumor celî lines. The resuits showed that both ceil lines do not express detectable

levels of IGF-1 mRNA. Similar levels of mRNA for TGFa are detected in both ce11 lines.

M-27 ceiis expresse simiificantly higher levels of mRNA for EGF, EGF receptor, PDGFa

and PDGF receptor bha in than tumor H-59 cells. In addition, ceil migration in response

to IGF-1 was measured and the d t s indicated that IGF-1 is chernotactic for tumor H-

59 but not M-27 celis. A western blot analysis with antibodies to phosphotyrosine

showed that foliowing IGF-I bindiag several proteins are phosphorylated in H-59 but

not in M-27 cells. Finally, a DNA hgmentaîion assay showed that 8 5 9 ceils expressing

IGF-IR antisense RNA but not wild-type ceils undergo apoptosis when incubated in

serum -fkee medium supplemented with IGF-1 only.

Reagents and eDNA probes Recombinant human [GF-1 and the monoclonal antibody

0 to phosphotyrosine wem purchascd h m UBI (Lake Placid, NY). Rat fibronectin was

Li Long. Reguhtion of Tumof Ceil IIIV(U~OR anà . k t a r a s u by rtic Typz I f~uu i in - f i& Growth Factor Recrptor (IGF-I R)

0 from Sigma (St. Louis, MO). The [GF-I cDNA probe was a kind gift fiom Dr. Henry G-

Friesen (Department of Physiology, University of Manitoba, Canada), cDNA probes for

IGF-I (437), EGF (438), EGFR (439), TGFa (440), PDGFa (Ml), and PDGF-RB (442)

were purchased fiom the ATCC (Rockville, MD). The alkaline phosphatase-conjugated

goat anti-mouse antibody was fkom BIOKAN Scientinc (Mississauga, Ont ).

Northern blot anaiysis: Cellular RNA was extracted by the phenol-chloroform method

of Chomczynski and Sacchi (443). The RNA species were rcsolved by electrophoresis on

1.2 % agarose gels containhg 3.7% formaidehyde (444). Approximately 30pg of total

RNA were loaded ont0 each lane. The htionated RNA species were transferred ont0

charged nylon membranes (GeneScreen Plus, New England Nuclear Research Products,

Boston, MA) by the capiliary blotting method me cDNA probes were radiolabeled by

0 the random primer method of Feinberg and Vogelstein (445). The blots were incubated

with the radiolabekd probes for 24 h at 37OC, washed and subjected to autoraàiography.

The relative amounts of the mRNA &pts were analyzd by laser densitometry ushg

an LKB Bromma Ultroscan XI, Enhanceà Laser Densitometer and normalized relative to

the 18s rRNA.

Boyden chamber migration usay: Eight p polyvioyl-pyrrolidine-ke filtecs

(Nucleopore Corp., Pleawinton, CA) were coated for 15 min with rat fibronectin diluted

in phosphate-buffered saline (without calcium or magnesiimi) to a final concentration of

13.3 pg/mi. The wated filters were placed on a 48 weii migration chamber (Neuroprobe,

Cabin John, MD) containhg dinerent dilutions of IGF-I in serum fhe RPMI and the

0 migration chamber sealed. To the upper wells 15,000 tumor cells in 50 pl of RPMI were

added and the chamber incubated at 37OC for 4 h in a 5% CO2 atrnosphere. The side of

the filter onto which the cells were loaded was then scraped fiee of cells, the migrating

cells fixed in f o d i n for 45 min_ the filters washed in phosphate-bufEered saline and

stained overnight in GU'S triple strrngth hematoxyh (Polyscienca, Warrington, PA)

and then washed again in phosphate-buffered caline, and mounted in glycerol. AU the

ceils within the perimeter of each weli were counted using a Light microscope.

Detection of tyrosincphospho ylihd proteins by Watern blotting. Subconfluent

cultures of tuxnor cells were washed twice with serum-ike RPMI and incubated in serum-

fkee RPMI for 18-24 h. The medium was removed, and the ceiIs supplemented with h s h

serum-6ree medium containing 50 nghl IGF-1 and incubateci at 37OC for different

intervals indicated in the te- The medium was removed, the ceil monolayers quickly

lysed using the RIPA lysis b e e r (50 mM Tris, pH7.5, 1 50 m . NaCl, 1 % Triton X- 100,

1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM dithiothreitol, 100 p M sodium

orthovanadate) containhg 1% Trasylol and 20 p M leupeptin. The lysed celis were

tramferred to wld mictofhge tubes, vortexed, incubated on ice for 10 min and then

centrifuged for 10 min to remove nuclei and ceil debris. The ce11 lysates were aliquoted

and stored at -20°C until used. For analysis the sa&les were separated by electrophoresis

using 7.5% polyacrylarnide gels under reducing conditions. The separated proteins were

transblotted onto a nitrocellulose membrane, non specific protein binding blocked with

blocking buffer (3% BSA and 5% skim m2.k powder in 1M Tris pH 8.0) and the

a membranes Uicubated first with an a n t i i y to phosphotyrosïne ( 1 500) ovemight at 4OC

and then with a 1 :200 dilution o f an alkaline phosphatase conjugated second antibody for

mouse IgG. Protein bands were developed using the substrate NBT/BCIP (nitro blue

tetrazolium/5-brorno-4i:hlom-3 -indolylqhqhk) as we described previously (390).

DNA bgmentation rwsay. Tumor H-59, SS-2 and SA-9 cells (see chapter 4 and 6 )

were first cultureci in RPMI containhg 10% FCS. Confluent ceiis (8x109 were washed

three times with serum the medium and re-cdttired with serum Eree medium and IGF-1

(1Ong/ml, UBI Inc., Laite Placid, NY) for 72 h. Ceils were then washed with ice-cold

PBS, centfigeci at 1200 rpm for 10 min, and resuspended in Lysis bUner consisting of 10

mM Tris-HC1 (pH 8.0), 10 mM EDTA, 0.5% SDS, 100 pdmi Rnase A, and 100 pghl

proteinase K (Sigma). After incubation at 37OC for 16 h, samples were extracted twice

with pheno1:chlorofonn (v/v) and then precipitated ovemight at -20°C by adding 1 :10

(v/v) sodium acetate (3 M, pH 5.2) and 2 volumes of absolute ethawl. DNA was then

peileted by centrîfiigation at 13,000 x g for 10 min, washed with 1 volume of 70%

ethanol, and air dried. DNA was fe~u~pe~lded in 10 mM Tris-HCl - 1 m M EDTA b a e r

(pH 8.0) containing the loading buffer (50% glycetol-0.05% bromophenol blue-0.05 %

xylane cyan01 FF). The samples were electmphoresed on 1.2 % agarose slab gels

containing ethidium bromide and the DNA bands visualized with a UV transilluminator.

Dinerential expression of growth factors and receptors in 8-59 and M-27 ceUs.

The results of Northern blot analyses of growth factors and receptors expressed by these

celis can be sumrnarized as follows:

IGF-1: Anaiysis of mRNA extracted h m mMae hepaîocytes (the positive control)

revealed two traascripts of 7.0 and 1.5 kb (Fig 701% lane Hep.). Neither of these

transcripts codd be daodod in Northern blots of H-59 and M-27 (Fig 7-la).

EGF and EGFR : Two EGF transcripts of 6.4 and 4.0 kb were detected in mRNA

extracted h m M-27 but not h m H-59 ceUs (Fig 7-lb). Similarly, in M-27 but not in

8 5 9 blots probed with an EGFR cDNA, three transcripts of 10.2, 5.6 and 2.5 kb were

detected @ig 7- 1 c).

TGFa: A major 5 kb and two additional (1.5 and 0.8 kb respectively) transcripts were

detected in extracts of both celi lines using a buman TGFa cDNA probe (Fig 7- Id).

PDGFa and PDGF receptor bhain:: M-27 express significantly higher levels of mRNA

transcnpts for PDGFa and PDGF receptor bchain than H-59 cells (Fig 7-le and f ). The

results are also summarized in table 7-1.

Table 7-1. Dinerential expression of growth factors rnd meptorr in 8-59 and M-

27 ceus.

EGF EGF-R TGFa PDGFa PDGF-R$

Li Long. Reguhtion of Timor Cell Inwion a d Metas~asis éy the Type I Ikuiin-like Growrh Factor Recvpror (IGF-IR)

a IGF-1 is chernotactic for Li-59 but not M-27 ceîis. Tumor cell ability to migrate toward

an IGF-1 gradient was assayed in a modined Boyden multiwell chernotaxis chamber. As

shown in Fig 7-2, M-27 cells migrated poorly in response to [GF-1, while H-59 celis

migrated in a dose-dependent manner.

IGF-1- induced protein tyrosine phosphorylation in tumor H-59 ce&. IGF-1 induced

tyrosine phosphorylation was d y z e d by the Western blot assay using a monoclonal

anti-phosphotyrosine antibody. Three phosphotyrosine bands of Mi 150,000- I60,OOo

@p 1 50- 16O), A4k l90,OOO @p 190) and 76,Oûû-8 1,000 (pp76-8 1) codd be detected in

H-59 extracts foliowing addition of IGF-1. Phosphorylation of the pp190 protein was

apparent as eady as 10 sec foiiowing IGF-1 addition, reached maximal intensity at 30

seconds and began to niminish after 5 min. In the absence of IGF-1, only weak

phosphotymsine bands were seen at the 150-160 and 7681 kDa range. foilowing the

addition of IGF-1, the intensiîy of these bands increased reaching a plateau after 60 sec.

H-59 ceb expraming IGF-IR antheme RNA unnot be rescueà h m apoptosis by

IGF-1. During apoptosis, 10s of membrane integrity is typicaliy preceded by chromatin

condensation and internucleosomal cleavage of genomic DNA (446). When cultured in

senun fke medium supplemented with XGF-X for three days, the XGF-IR antisense cDNA

transfected H-59 clone SA09 ceiis contained large amounts of low molecular weight

DNA, which produced a characteristic "ladder" on agarose gels (Fig 7-4, lane 1). The

degraded DNA was present in oligomen that were multiples of approximately 180-200

base pairs suggesting internucleosomal cleavage. In contrast, this DNA fÎagmentation

pattern was not obxrved in B59 or sense transfected SS-2 ceus cultured under the same

conditions (Fig 74, lane 3 and 2 respectively) indicatiug that in these ceiis the activated

IGF-IR could protect the ceUs h m apoptosis.

Li Long. Replation of Timor CeAI Inwuon and .Ue~cutasÛ by r k Tvpe I Insufin-Iikz Growth Factor Receptor (IGF-IR)

The data on the differentiai expression of growth factors/receptors in the tumor Lines add

another level of complexity to the anaiysis of cellular and molecular factors involved in

target organ specificity in our turnor model. Growth factors cause celis in the Go phase

to enter and pmgress through the ce11 cycle. The quiescent ce11 m u t first advance into the

G1 phase of the celi cycle by '%ompetence" factors such as PDGF. This factor and its

receptor are e x p d in both H-59 and M-27 cells although the level of expression is 5-

2-fold higher for PDGFa and PDGF-RB respedvely, in M-27 as compared to H-59 celis

(Fig 7-le and f). Once celis enter G1 they can becorne committed to DNA synthesis

under the infiuence of 'cprogression" factors such as IGF-1 and EGF provided they

express the respective receptors. Expression of i'progressi~n'' fators and their receptoa

in H-59 and M-27 cells are sigdicantly different. Namely, tumor H-59 celis express

significantly higher levels of the IGF-1 receptor than M-27 ceils (Please see Chapter IV)

whereas the latter express sigaificantly higher levels of EGF and EGF receptor, indicating

that the two d s may utilize different progression mechanisms. Interestingly, neither

tumor expresses detectable levels of IGF-1, suggcstiag that exogenous IGF-1 is required

to induce IGF-LR dependent "progression", particularly for H-59 d i s . These results raise

the possibility that the differences in the patterns of metastasis of these sublines iq vÏvo

are affecteci by their distinct repertoks of growth factorts) and receptor(s) although the

mechanisrn is not entirely clear.

IGF-I is also a ce11 migration factor (407, 410). As shown in Fig 7-2, H-59 migrated

significantiy better than M-27 cells towards an IGF-1 gradiant probably as a result of the

increased number of binding sites on these cells. As the liver is the major site of IGF-1

production (Fig 7-la), it is conceivable that IGF-1 plays a role in directing H-59 to the

liver as well as supporting H-59 growth in the liver once the cells are arrested in this

a organ. Tumor M-27 cells do not migrate in responce to IGF-1 and therefore may have a

preference for the tung where high levels of PDGF have been reported (20).

Many growth factors stimulate cellular mitogenesis by interaction with a family of cell-

surface receptors that possess an intrinsic, Ligand-sensitive, protein tyrosine kinase

activity in the receptor cytoplasmic domain (447). The critical events foilowing the

binding of IGF-1 to the IGF-1 receptor are the activation of the intrinsic tyrosine kinase

within the intracelluiar domain leading to tyrosine autophosphorylation and subsequent

phosphorylation of tyrosines on other cytoplasmic substrates (448). A major substrate of

the insulin receptor is W-1, a cytoplasmic protein which migrates as a 160-185 kDa

band in SDS-PAGE (299). Following insulin/IGF-1 stimulation, IRS-1 is rapidly

phosphorylated on sultiple tyrosines (299). This r d t s in docking of several SH2 domain

proteins, including: the p85 subunit of PI Ikinase (449), an upstream element in insulin-

stimdated glucose transport and activation of p70 S6 kinase (450); Grb2, an adapter

molecule linking IRS-1 to activation of Ras and mitogm-activateci protein (MAP) kinase

(45 1); and the tyrosine phophatase SHPTP2 (307). IGF-1 receptor can also phosphorylate

other cytoplasmic proteins. These include Shc, a cytoplasmic protein with reported MW

of 46, 52, and 66 kDa which biads to Grb-2 (308, 452), a p62 protein which associates

with Ras-GAP (453). and a 55-60-kDa protein which associates with PI 3-kinase (454).

Recentiy, a Mr 180,000 protein desiguated IRS-2, has been rrported which is tyrosine-

phosphorylated within 1 min of insuiin stimulation, binds to PI 3-kinase and is not

recognized by anti-IRS-1 antibodies (455). The substrates of IGF-IR in H-59 cells

following activation by IGF-1 need to be firrther investigated but the protein bands

identified by Western blotting with anti-phosphotyrosine antibodies comspond to IRS-1

(pp- 1 50- 1 6O), IRS-2 (pp 1 90) and subunit of PI3 kinase @p76-8 1) in their estimated MW.

Numerous reports have recently identifieci IGF-1 as an auti-apoptosis factor. IGF-I was

show to maintain the viability of non-proliferating celis in culture, notably neurones

(332,408,456458)- IGF-1 can also rescue neuronal celis in culture h m ischemia (459)

and cerebeiia granule cells h m potassium shock (460,461). Evan and coworkers have

demonstrated that IGF-I can prevent c-myc-induai apoptosis in rat- 1 fibmblasts (333).

Activation of the IGF-1 meptor couid dso protects BALB/c 3T3 fibroblasts from

apoptosis induced by serum deprivation or exposure to the topoisornerase inhibitor

etoposide (335, 462, 463). In other studies, IGF-1 and the IGF-IR were shown to be

required for swival of cuitured hmmtopoietic ceiis aAer tmphic fmor withdrawal(334)

and to block the death of a variety of tumot ceii Lines cultured for short term in vivo (33 1,

336). One general conclusion that emerges h m these varius observations in that IGF

action can prevent the premature death of many ceii types, a conclusion consistent with

the rnarked cellular hypoplasia in tissues of mice lacking a fimctioning XGF-Et (329).

b. EGF h

d. TGFa h f ;

EGF-R

10.2 kb

Fig 7-1. Cornparison of expression of gmwth factors and p w t h factor receptor mRNA

transcripts in 8 5 9 and M-27 ceiis. a IGF-1; b. EGF; c. EGF-R; d. TGFa; e. PDGFa; f.

PDGFRQ. The sizes of major mRNA bands are shown on the left. The fïiter was also

hybridized with a cyclophilin cDNA probe (panel b - f )

Fig 7-2

Fig 7-2. Migration of tumor ceiis in response to IGF-1. Ho59 (e) and M-27 (m) cells were anaiyzed as mean (f SD) of triplicaîe weiis. The ôars qresent standard deviation of the mean.

Li Long. Regdation of T i o r C d Inwrioi, and ~ ~ ~ P I U by the Typr I fkmIin-likc Growtli Factor Rcceptw (IGF-IR)

Fig 7-3. Westem blot Analysis of IOF-1 -inducecl tyrosine phosphorylation. Smim-

starved H-59 celis wm incubated with 50 n g h i of IGF-1 at 37OC for the indicated

intervals and immediately lyseci. Anti-phosphotymsine Mab was used at a dilution of

1:500. The positions of M W standards are marked on the le& The estimated molecular

weights of phosphorylated protein are marked on the ri@.

Fig 74. IGF-1 prevents DNA fragmentation in tumor H-59 and SS-2 ceiis. Tumor cells

(approximately 8x10~ ) were lysed, and the DNA was isolated and electrophoresed as

described in "Materiais and Methods''. A DNA ladder (M) was run as a molecular size

standard.

Chapter Vlll

Discussion

Li Long. Regufarian of Tumor Cefl f ~ i m and M'tastp~ir by t/ru Tjpe I f'fin-lilr Growth Factor Rrccptor (fGF-f R)

The evidence described demonstrates that the insulin-like growth factor I receptor is

involved in carcinoma growth, invasion and metastasis to the liver. These results provide

the fi& indication that the IGF-MGF4 complex play a role in preferential homing of

cancer cells to selected target organs and in regulating cellular invasion through its role in

regulating expression of the Mi 72,000 type IV collagenase - a major mediator of ECM

proteolysis.

8.1 Dinetentid erprruion of p w t h factorr and recepton: tumor heterogeneity.

Metastasis, the spread of cells b m the primary neoplasm to distant sites and their growth

there, contributes to the death of most cancer patients. The two major reasons for the

failure to treat metastases are the anatomid location of the metastatic lesions and the

biologic heterogeneity of ceils in primary and secondary neoplasms. This heterogeneity is

manifest in a wide range of genetic, biochemical, immunological, and biological

characteristics including ceII sdace receptors, enzymes, karyotypes, c d morphology,

growth properties, sensitivity to various therapeutic agents, and the ability to invade and

metastasize (464). A century ago. Paget questioned whether the distribution of metastases

was random and therefore analyzed a large number of autopsy records of women with

breast cancer. The noorandom pattern of visceral breast cancer metastases suggested to

Paget that the process was not due to chance, but rather th certain favored tumor cells

(the "seed") had a specinc e t y for growth in the milieu provided by certain organs

Li Long. Regulafiom of Tmor Cell fnvasion ami d c k ~ a s ~ i s by the Typr I fnsutin-like Growth factor âeceptor (IGF-IR)

(the "soil"). Metastases resulted only when the "seed and the soir were compatible (1).

This concept has gained wide acceptance and currently is based on three principles:

1. Neoplasms are heterogeneous in respect to biologic and metastatic properties;

2. The process of metastasis is not random, but consists of a series of iinked, sequential

steps that must be completed by tumor cells if a metastasis is to develop;

3. The successfùl establishment of metastasis depends on the outcome of interaction

between the multiple metastatic celis and the cliffixent target organ microenvironment.

Previous studies in our laboratory revealed that the Lewis lung carcinoma sublines H-59

and M-27 différ in a range of phenotypes including ceii adhesion (388-391.46s) and the

expression of ECM degrading proteinases (392) ali of which may contribute to their

organ selective pattems of metastasis. These p h e n ~ ~ i c difference may be related to the

divergent expression of several growth factors and their receptors in these ceiis as

described in the present study. The molecular mechanisms which underlie the

development of tumor heterogeneity are divene. They rnay reflect genetic events such as

mutations chromasomal deletions or transtocations which alter gene expression.

Aitematively altered gene expression rnay be due to epigenetic mechanisms such as DNA

methylation and demethylation, processes which can be induced environmentally and

developmentally (466,467). The mechanism which caused the development of the Lewis

lung carcinoma sublines with distinct invasive/metastatic properties used in this study

are not presently clear.

Li Long. Rrgulation of Tiunor Ceii Inwsion ami .Uemrusis by the T ~ p e I lnnriui-like Growrh Facror Recrpcor fIGF-I R)

The overexpression of IGF-IR in tumor H-59 cells (seed) may be a major mechanism

regulating their preferential metastasis to the liver (soil) since the latter is a major sources

of IGF-1 production (Chapter W. Fig la, and see ref. (20,468). This is supported by our

fmding that M-27 cells transfected with full length IGF-IR cDNA acquired an incfea~ed

liver-colonizing potential (Chapter V). Neither H-59 nor M-27 cells express detectable

IGF-1 levels (Chapter W, Fig la). The activation of the IGF-iR in these cells therefore

relies on an exogenous source of IGF-1 rendering the liver a suitable target organ. IGF-1

has also been identifieci as a chernotactic andior motility factor for many normal or

malignant cells including endotheliai celis (469), keratinocytes (470). osteoblasts (471),

smooth muscle ceii (261), melanoma (84), breast carcinoma (472), bladder and ovarian

carcinomas (472). In our studies it was found that IGF-1 is a chemoattractant for H-59 but

not for M-27 ceiis (Chapter W, Fig 2). It is conceivable that the chernotactic effect of

IGF-1 also plays a role in selcctively directing tumor migration towards organs with high

local concentrations of this factor. In addition, the mitogenic (Chapter IV and V), anti-

apoptotic (33 1, 335, 336, 409) and invasion-inducing effect of IGF-1 (Chapter VI)

probably contribute to its role in promoting target organ colonization. A similar

interpretation can also be applied to lymph node metastasis of 8 5 9 cells since IGF-1 is

aiso produced in the lymph nodes (20). The influence of the differential expression of

other growth factors and receptors on invasion and metastasis in our tumor mode1

requises M e r investigation.

8.2 The rote of the IGF-IR/IGF-1 compkx in cancer invasion and metastrsis

Li Long. Re&ation of Tmor Cell fn\wlion and .Hokastaris by I/w Tjpc 1 f~~~IUi--liitc Growrli Facroc Reccpror (lGF-C R)

0 The process of metastasis is a cascade of Linked sequential steps involving multiple host-

tumor interactions. This complex process requires the cells to eriter into the circulation,

arrest at a distant vascular bed, exüavasate into the orgau interstitiun and patenchyma,

and proliferate as a secondary colony. increased expression of IGF-IR andor IGF-1 has

k e n noted in a wide range of tumor types including carciinornas of the lung, breast,

thyroid, gastrointestinal tract, iîver, pancreas and kidney, and neuroendocrine tumors

(339). In vitro stuàîes have irnplicated this receptor in cellular transformation (3 13, 327,

328), proiifèration (326, 330), ceU protection h m apoptosis (331, 335, 336) and

migration (84,261,407,410). Ilan and CO-workers have found that dsense RNA to the

IGF-IR could inhibit tumor growth and prevent invasion rat prostate cancer cells vivo

(473). Ioterestingiy, a signiscant reduction in expression of both tPA and uPA which play

a important rolc in matrix degredatîon o c c d in IGF-IR antisense transfected celis

(473).

As we have discussed in Chapter 6, IGF-I has been implicated in transcriptional

regulation of various genes including the early response genes fus (304,427,428) andjun

(429), cell cycle intermediates such as cyclin-Dl and cdc2 (430) and recently, the

urokinase plasminogen activator Uihibitor PM4 (43 1). IGF-1 has also been implicated in

the regulation of the synthesis of ECM pmteins such as collagen (432) and proteoglycans .

(433). As IGF-I has been shown to enhance steady state mRNA levels through a direct

effect on gene transcription or by pst-transcriptional modification of m.RNA, which

e stabilize the message (43 l), the precke molecular mechanism involved in regulation of

MMP-2 synthesis rem- to be elucidated.

Multiple signal transduction pathways can be hnned on in response to ligand binding by

IGF-IR. They include a tyrosine phosphorylation cascade involving IRS-1 (insulin

receptor substrate-l), Shc, Grb-2 and mSOS which are Linked to the ras signaling

pathway (reviewed in ref. (S9), activation of phospholipase C and the accumulation of

IP3. While the link between these signahg intermediates and MMP-2 synthesis remains

to be elucidated, it is of interest to note that IRS-1 was recently found to be associated

with the integrin vitronecth receptor a& (434), a modulator of MMP-2 synthesis which

was also recently identifieci as a cell surface receptor for MMP-2 (435). The signal

transduction pathways activated by IGF-IR and a& appear therefore to converge with

the iikely result of signal amplincation and an enhancement of the proteolytic and

invasive capabilities of the ceii. In a recent report, it was shown that the inhibition of

TGF-1 B receptor-mediateci calcium influx blocked the activation of MMP-2 transcription

in three different human tumor hes (421). This suggests that calcium mobiiization is a

critical component of TGF-lp-mediated activation of MMP-2 synthesis. A s Ligand

binding by IGF-IR is known to induce to a rapid accumulation of IP3, resulting in

increased cytoplasmic leveb of fÏee calcium (436). it may be one mechanisms for IGF-I-

mediated regulation of MMP-2.

Li Long. &guIation of T-of Ce4 i . . i o n und .Uecprtosir & r i te T m I In~Iin-Iike Growrh Factor Receptor (IGF-IR)

While selective H-59 metastasis to the liver may be regulated by IGF-1, the mechanisms

which regulate M-27 metastasis to the lung are not presently entirely ciear. M-27 cells

express significantly higher levels of EGF, PDGF and the respectiire recptors than H-59

cells (Chapter W, Fig 7-1). Overexpression of IGF-IR in these ceiis did not result in a

significant change in either tumongenicity, meanad as the incidence and growth rate of

subcutaneous tumors, or the potential to colonize the lung following i.v. inoculation

suggesthg that the growth of these ce& in vivo in these sites may be regulated

predominantly by autocrine mechanisrno mediateci by growth factor(s) such as PDGF and

EGF. In addition, M-27 ceUs produce significantly higher leveis of urokhase-type

plasminogen activator @PA) and cathepsin B than tumor H-59 celis and it is wnceivable

that the differences in the types of basement membrane d e m g proteinases released by

these tumor cells are also a factor in their distinct patterns of dissemination. Other

factor@) such as transfh have been identifiai as regdators of lung metastases

formation and they may also play a d e in our system (474). The hding that the major

effea of increased IGF-IR expression on M-27 c d growth ip v i v ~ was a rnarked increase

in their liver-coloniring potentid to levels normally observed with carcinoma H-59

supports the conclusion that IGF-I plays an important role in the pmcess of liver

metastasis.

The involvement of IGF-IR in the mgdation of type IV collagenase synthesis may e t

not only the invasive potential of the ceiis but also cellular proliferation. Recent studies

by Dr. Fowlkes and colleagues have demo~l~trated that IGF binding proteins 3 and 5

(IGFBP3 and 5) are processed by MMPs (MMP-2 and MMP-9) both in vitro and

suggesting a role for MMPs in regulating semm and tissue IGF bioavailability (52, 53,

289). Taken together wîth our data, this is suggestive of a possible IGF-UIGF-IR:MMP

:IGFBPs loop regulating IGF-1 function, namely, IGF-1 can upregdate the expression of

MMP-2 (Chapter VI), active MMP-2 in tum is involved in the processing of IGF

binding proteins (52, 53,-289) which can modulate the interaction of the IGFs with theu

receptors (284). A schematic illustration of the proposed IGF-MGF-IR:MMPs:IGFBPs'

loop is shown in Fig. 8-1.

modulation of IGFaGF-CR interaction

upctguiation ? transcription ? mRNA stability

Li Long. Regukz~ion of Tumor Ce fi I l l ~ ~ ~ i o n and Mkku&tsïs by clir Typc I IlLnJWik Gmwh Facror Rrcepror (fGF-/ R)

Fig. 8-1. The schematic Uustration of proposeci IGF-VIGF-1R:MMPs:IGFBPs' Loop

Li Long. Replation of Tùmor Cell Invasion Md ,UerartasU d u f farulin-lik Growih Factor Reccpror (IGF-IR)

Cellular invasion depends on adhesion as well as proteolytic modification of the

extracellular matrix and an association between the expression and fùnction of adhesion

receptor; and ECM-degrading proteinases has been noted in ciiffernt systems (122, 143,

475). For example, Brooks et ai have recently reported on the colocalization of MMP-2

and the vitronectin receptor integrin a,,& on angiogenic blood vessels and melanoma

cells Jn vivo. Expression of q P 3 on cultureci melawma cells was necessary for binding of

MMP-2 in a proteolyticaIiy active form, facilitating cell-mediateci collagen degradation

(435). In our laboratory, a fùnctional and rcguiatory link between a& and the urokinase

-type plasminogen activator receptor @PAR) has been identifiecl in metastatic melauoma

cells (189). Interestingiy. Jones et al have recently reported that ligand-dependent

. activation of a$3 is necessary for IGF-1-mediated stimulation of ce11 migration (410),

providing a mechanism for coordination of ceil adhesion, migration, invasion and growth

through molecular cross-talk. The expression and the d e of the vitronecth receptor

in our tumor mode1 have not been studied. A M 64,000 plasma membrane glycoprotein

has k e n icientifïed as an adhesion molecule mediating the speciiic interaction between

tumor H-59 cells and hepatocytes, an important step in liver colonipltion by tuxnor H-59

cells (390.391). The relationship between tbis d e s i o n molecule, the IGF-IR and matrix

degrading proteinases, if any, remains to be determineci.

Recent insight gained into the molecular mechanisrns underlyiag the pmcess of ceIl

migration and invasion has led to the identification of two major categories of cellular

targets for therapeutic intervention. The k t category includes celi M a c e and secmted

Li Long. Rrgufation of Tiunor CeII fmasiion anâ lUetas~asis tk Tjlpc I Insuiin-like Growth Factor Rcceprar ffGF-f R)

a proteins such as adhesion receptors, degradative eazymes and their inhibitors, growth

factordreceptors and motility stimuiating cytokines (1 1, 157, 199, 3 70, 476-478). The

second category is defined as intracelîular regulatory proteins which mediate signal

transduction mechanisms such as G-pmteins, and tyrosine kinases (342,479-482). Many

studies indicate that the IGF-VIGF-IR complex and metalloproteinases may provide

effective targets for cancer therapy (331,342,343,345-347,394,417,473,483486). The

present hdings confirm and extend previous findings on the critical role of the IGF-

VIGF-IR system in cancer celi growth, invasion and me-. Together they suggest

that the IGF-VIGF-IR system codd provide a target for anti-metastatic therapy.

8 3 Suggestions for Fuhuc Rcscarch

The following aspects requin M e r investigation:

1. Elucidating the signalhg pathways linking MMP-2 transcription to IGF-1 receptor.

2. Analyze the relaîionsbip between IGFBP production and MMP-2 synthesis and

function in H-59 and M-27 cells.

3. Gene therapy of metastatic tumors by transduction with a viral vector expresshg IGF-

1 R antisense DNA.

4. Mutational analysis of the IGF-1 receptor to identiSr receptor domains critical for its

diverse f'unctions.

Li Long. Regubtion of Twnor Ceff Invasion and Me~cutasu by the Tjpc C I~ufin- l ik Growth Factor Receptor UGF-1 R)

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