ROLE OF CELL ADHESION MOLECULES IN MELANOMA

TRANSENDOTHELIAL MIGRATION

Ning Chen

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Biochemistry

University of Toronto

@Copyright by Ning Chen 2001

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Role of ce11 adhesion molecules in melanoma transendothelial migration

Ning Chen

Master of Science

Department of Biochemistry

University of Toronto

2001

ABSTRACT

Transendothelid migration is an important step in tumor ce11 metastasis. Using

an in vitro assay, 1 demonstrated the involvement of N-cadherin in melanoma

transendothelial migration. irnrnunofluorescence labeling studies revealed that N-

cadherin becarne concentrated in the heterotypic contacts. inhibition of N-cadherin

expression in WM239 cells led to -30% inhibition on melanoma transendothelial

migration. In contrat to adherens junctions, there was no preferential association of P-

catenin with N-cadherin in the heterotypic contacts where of N-cadherin was enriched.

The potential involvement of gap junctions during transendothelial migration was dso

examined by irnrnunofluorescence staining. Inhibition of gap junction function using i -

heptanol resulted in a substantial reduction in melanoma ce11 transmigration. These

results suggest that N-cadherin may provide weak adhesive interactions between the two

ce11 types during the transmigration process and that gap junctions may provide channels

for the transfer of signais that induce the retraction of endothelid cells.

Acknowledgments

Dunng my study in Dr. Siu's laboratory, the most important achievement for me

was that 1 learned to think as a scientist. Dr. Siu has always encouraged me to formulate

ideas using my own brain, to observe though my own eyes. 1 believe that I will benefit

from this for my whole life. 1 would like to take this opponunity to thank Dr. Siu, my

supervisor, for his teaching, helphlness and support.

1 would also like to thank my graduate cornmittee members. Dr. Sodek and Dr.

Hannigan. Over my graduate studies, they have provided key suggestions that have

helped irnprove my expenments and thesis.

People in Dr. Siu's laboratory have been very helpful to me. 1 want to thank al1 of

them, past and present. 1 have had wonderful discussions on both scientific and non-

scientific topics with them.

Lastly and dearest to me. 1 thank rny wife, Wenbo, for her support during my

Master's study. She has commuted between Waterloo and Toronto every week. She

takes care of me in life and dways gives me vaiuable encouragement and criticism,

whichever is in need.

Table of Contents

Abstrâct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table of Contents . .. . .. .. .. . . . . . . . . . . .. .. . .. . . . . .. . .. .. . . . . . . . . . . .. . .. . . .. . . . . . . . . - .. . .. . . . ....

List of Figures and Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

List of Abbreviations ... . .... .. ........ ... ... ... ... ... .... ... .... . .... .. .. -.. . .... .... .. . . .. ..

Chapter 1: Introduction ... ... ... . . .. ........... . .... . ...... ... . .... . ... ....... . .. ... . .. .. .,

Chapter 2: Materiais and Methods . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 3: Results ...... .. . . . ... . .. . .. .. . .. . . . . .. . .. . . . . . . . . .. . . .. . . ... .. . . . ... . . . . . . . . . . . . . .. 42

Chapter 4: Discussion ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

, . 11

. . . 111

i v

v

vii

1

33

List of Figures and Tables

C hapter 1: Introduction

Figure 1 Tumor metastasis

Figure 2 Composition of classical cadherin complex

Figure 3 Inter-endothelial junctions

Figure 4 The in vitro mode1 of melanoma transendothelial migration

Chapter 2: Materials and Methods

Table 1 Melanoma ce11 lines used in expenments

Figure 5 Structure of the N-cadherin antisense plasmid, pBSpacA'

Chapter 3: Results

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Cadhenns expressed in melanoma cells and HMVEC 52

Lncrease of levels of N-cadherin during melanoma transmigration 53

Localization of N-cadherin in the heterotypic contact regions 55

E ~ c h m e n t of N-cadherin in the heterotypic contacts: early stages 57

Enrichment of N-cadhenn in the heterotypic contacts: late stages 59

WM35 cells cannot form N-cadherin contacts efficiently with HMVEC 6 1

Expression of an N-cadhenn variant in W M 3 5 cells 63

Effect of the commercial antisense oligonucleotides on N-cadherin expression 64

Inhibition of N-cadherin in WM239 cells using antisense construct 65

Effect of reduction in N-cadhenn on melanoma transmigration 67

Lack of D-catenin in the N-cadherin mediated heterotypic contact regions 68

Figure 17 Changes in the profiles of proteins associated with N-cadhenn

Figure 18 [mmunolocaiization of gap junctions in the heterotypic contacts

Figure 19 Inhibition of melanoma transendothelial migration by 1 -heptano1

Chapter 4: Discussion

Figure 20 Switching of E-cadherin to N-cadhenn during melanoma progression

Figure 2 1 A working mode1 of the role of N-cadherin in metastasis

List of Abbreviations

12(S)-HETE M C

bp CAM

EC domain

E-cadherin

EGF

FBS m c GlcNAc-T / GnT

GPI HBSS

HMVEC IC AM

k kDa

rnAb

MALDI N-cadhenn

PAGE PBS

P-cadherin

PECAM

RGP SDS

sLeS

TNF-a VCAM

VE-cadherin

VGP VLA

12(S)-hydroxyeicosatetraenoic acid

Adenornatous polyposis coli

Base pair

Cell adhesion molecule

Extracelluiar domain

Epithelial-cadherin

Epidermai growth factor

Fetal bovine serum

Fluorescein isothiocyanates

B-N-acety lglucosarninyltransferase-T

Gl ycos y lphosphatidylinositol

Hank's buffered saline solution

Hurnan rnicrovascular endothelial ce11

Intercellular adhesion molecule

Imrnunoglobulin

Kilodalton

Monoclonal antibody

Matrix-assisted laser desorption/ionization

Neural-cadherin

Polyacrylarnide Gel Electrophoresis

Phosphate-buffered saline

Placental-cadherin

Platelet endothelial ce11 adhesion molecule

Radial growth phase

Sodium dodecyl sulfate

Sialyl Lewis"

Tumor necrosis factor a Vascular ce11 adhesion molecule

Vascular endothelial-cadherin

Vertical growth phase

Very late antigen

vii

Chapter 1 : Introduction

1. Overview of Cancer Metastasis

The ability of malignant cells to develop secondary tumors in distant organs is the

most dangerous aspect of cancer. Research in recent years has revealed that the

establishment of metastases is the result of a series of complex interactions between the

cancer cells and their microenvironment (Nicolson, 1988; Brodt, 1996; Orr et al., 2000).

Blood and lymphatic vessels are major anaiornical pathways for cancer

metastasis. The formation of remote metastases includes several interlinked steps.

involving a variety of ce11 adhesive interactions between cancer cells and endothelial cells

(Fig. 1). Cancer cells first need to detach form the pnmary foci before they invade into

surrounding tissues. Then they may transmigrate through blood or lymphatic vessels.

travel in the circulatory system, and undergo exuavasation in rernote tissues

(Aznavoorian et al.. 1993). After the developrnent of secondary tumors, the newly-

formed lesions in a distant organ can themselves become the source of a new cycle of

dissemination. Tumor growth in secondary sites depends on many factors. Not dl cancer

cells can survive and proliferate into secondary tumon. It is common in expenmenial

models of metastasis to find organs in which there are no visible metastases, but

subsequent histological examination reveals many micrometastases consisting of

individual tumor colonies of very small diameter. Careful examination often reveals that

such tumors are avascular. They can survive for long periods of time without further

expansion, which is termed dormancy (Holmgren et al., 1995). It is possible that dormant,

avascular Nmors undergo apoptosis or programmed cell death before they c m establish

successfully in remote tissues (Zetter, 1998). With proper signals, such as the removal of

C Adhesion to endothelium in remote organ

b Intravasation

Secondary tumor

Fig. 1. Tumor metastasis. The formation of metastasis involves the following steps: (a)

Small pnmary tumors invade the local epitheliai basement membrane. (b) Blood vessels

provide a route of entry into the bloodstream and the tumor celis circulate until they (c)

attach specificdly to endothelid cells in the vessels (usually venues) of downstream organs.

(d) The tumor cells extravasate through the vessel wall and then migrate to sites proximal to

arterioles where their growh is enhanced. The proliferation of the primary tumors and

secondary tumoa requires the formation of new blood vessel or angiogenesis.

3

inhibition of angiogenesis, the dormant micrometastases can be activated and grow

rapidly (O'Reilly et ai., 1997).

While traveling in the circulatory system, tumor ceils interact with blood cells as

well as endotheliai cells. The interaction between tumor cells with endothelia has a

significant effect on the process of metastasis and the formation of secondary tumor.

Initial binding between tumor cells and endothelial cells may provide weak interaction.

This can iead to stable binding via ce11 adhesion molecules. such as integrins. Since the

endotheliai junctions contain a high concentration of various ce11 adhesion molecules

including VE-cadherin and PECAM- 1. extravasation will involve the dissolution of the

interendothelid adhesion complexes and/or the formation of new ce11 adhesion between

cancer cells and endothelial cells. Therefore, an undentanding of the molecular

mechanism, especially the ce11 adhesion molecules involved in maiignant cell

transendothelial migration is of both biological and therapeutical importance. The work

included in my thesis is focused on the possible involvement and functions of ce11

adhesion molecules in the transendothelial migration of melanoma cells.

2. Cell Adhesion Molecules

Cell adhesion molecules are very important proteins found on the ce11 surface.

Their functions include ce11 recognition, cell-ce11 adhesion, tissue morphology, signai

transduction and tumorigenesis (Gumbiner, 1996; Buckley et al., 1998). Ce11 adhesion

molecules are generdly separated into four major classes: selectins, integrins, members

of the immunoglobulin superfamily of ce11 adhesion molecules (Ig-CAM) and cadherins.

2.1. Selectins

Selectins are lectin-like molecules that bind carbohydrates in a ca2'-dependent

manner (Brandley et ai.. 1990). The three members of the selectin farnily, P-, E- and L-

selectin. were originaily discovered on platelets, endothelium and leukocyte, respectively,

and they are named accordingly. Each selectin molecule contains an amino terminal

~a"-dependent (C-type) lectin, an epidermal growth factor (EGF)-like domain. at least 2

short consensus repeats, a transmembrane domain and a cytoplasmic tail (Bevilacqua and

Nelson, 1993). Both the lectin and EGF domain are involved in ligand binding. Al1 three

selectins bind weakly to the ietrasaccharide sialyl Lewis" (sLex) (Vestweber and Blanks,

1999). Ligands of higher binding affinity have also been identified for each of the

selectin farnily. Members of selectin family play important roles in leukocyte homing and

may mediate leukocyte attachrnent to the endothelium (Bevilacqua et al.. 199 1 ; Mebius

and Watson, 1993). It has been shown that leukocytes initially start ro roll on the

endotheliurn which expresses molecules that promote transmigration. The rolling

behavior is largely supported by both L- and P-selectins [Dunon. 1996 #17 1 ; (Liu et al..

1998). Anti-L-selectin antibodies have been shown to prevent rolling of both monocytes

and neutrophils. It is of interest to note that some tumor cells utilize selectins in a sirnilx

fashion. Colon cancer cells have k e n found to undergo E-selectin mediated rolling on

endothelium (Giavazzi et al., 1993). Many carbohydrate ligands are unique to tumor

cells. For example, sialyl Lewis' (sLea), a variation of the sLeX. is not usually found on

leukocytes, but is expressed on tumor cells and will interact with al1 of the selectins

(Carlos and Harlan, 1994).

2.2. Integrins

Integins hnction as ce11 surface receptoa to many different ligands and may

mediate both cell-ceil and cell-matrix interactions (Hynes, 1987). Lntegrins are composed

of noncovaiently associated aB heterodimers (Varner and Cheresh, 1996a). Typically,

each subunit contains a large extracellular domain, a single transmembrane segment and

a short cytoplasrnic tail. As many as 16 a-subunits and 8 p-subunits have been identified.

With different combination of the ap subunits, 2 1 different ap painngs are known

(Chothia and Jones, 1997). Jntegnns bind not only to components of the extracellular

matrix, such as fibronectin, collagens and larninin, but also to other ce11 adhesion

rnolecules, such as VCAM-1 and ICAMs. The ligand binding of integnns is dependent

on divalent cations, including M ~ " , ca2+ and ~ n " (Mould et al., 1995). htegrins are

able to mediate signal transduction in response to both adhesive interactions outside the

ce11 (outside-in) and events inside the ce11 (inside-out) (Bumdge and Chrzanowska-

Wodnicka, 1996; Dedhar and Hannigan, 1996; Disatnik and Rando, 1999). The outside-

in signaling is mediated by the cytoplasrnic domains of a- and P- subunits. Inside-out

signal is possibly delivered through a conformational change in the extracellular region

between active and inactive States (Diamond and Springer, 1994). Integrins are involved

in the stable adhesion of leukocytes to the endothelium and transmigration. For example,

interaction between a& integrin and MAdCAM- 1 can facilitate both rolling and firm

adhesion of lymphocytes on high endothelid venues and P2 integrin is required for the

adhesion and the subsequent transmigration of lymphocytes (Carlos and Harlan, 1994).

The interaction between c@ 1 and VCAM- 1 is implicated in the extravasation of

lymphocytes (Fogler et al., 1996). Much work has been done on the expression and

function of various integins in many forms of cancer. Among different integrins, a role

of a& integrin in cancer progression has been implicated in many tumors (Vamer and

Cheresh, 1996b: Natali et al., 1997). In situ detection of a 4 3 3 and the p3 integrin subunit,

as well as the andysis of the expression level of indicate that the level of this

integrin increases with progressive stages of melanoma metastasis (Menmsky et al.,

1994).

2.3. Ig-CAMS

The immunoglobulin superfamily includes a large number of ce11 adhesion

molecules sharing similar structure (Williams and Barclay, 1988). They are found in cells

of the nervous, circulatory and immune systems at al1 stages of development

(Bmmmendorf and Rathjen, 1994). They al1 have one or more common Ig homology

unit, which is characterized by - 100 arnino acids in length and organized into the

antibody fold (Chothia and Jones, 1997). Another common motif is the fibronectin type

iE-like repeat expressed in tandem with the Ig-like domains. Ig-CAMs are able to

mediate homophilic and heterophilic interactions with a variety of ligands, with varying

~a"-dependence (Williams and Barclay, 1988). Recently, a new family of Ig-CAM has

been identified, which mediates sialic acid-dependent ce11 adhesion. thus termed as the

siaioadhesin family (Varki, 1997). Members of this family include CD22, CD33, myelin-

associated glycoprotein (MAG) and the Schwann ce11 myelin protein (SMP) (Schnaar et

al., 1 998). Many Ig-CAMs expressed in the immune system mediate heterotypic cell-ce11

binding via heterophilic binding mechanisms and they have been implicated in various

functions including lymphocyte recognition, horning and rolling, antigen presentation,

and T-ce11 activation (Carlos and Harlan. 1994). The major Ig superfamily members

involved in leukocyte diapedesis include VCAM- 1 (Fogler et al., 1996), platelet

endothehl ce11 adhesion molecule- 1 (PECAM- I or CD3 11, intercellular adhesion

rnoIecules- 1 (ICAM- 1), and the mucosal addresin (MAdCAM- 1) (Carlos and Harlan,

1994). Sorne Ig-CAMs are capable of heterophilic interactions with integrins and other Ig

superfamily memben. For example, ICAM- 1 interacts with LFA-1 (a$?) and PECAM- I

binds to (Fogler et al.. 1996). Several Ig-CAMs are found to be involved in cancer

progression and extravasation. For example, the carcinoembryonic antigen (CEA) is

widely used as a tumor marker (Tang and Honn, 1994b). This Ig ce11 adhesion molecule

cm promote colon cancer ce11 adherence to collagen. In various cancers. increased

ICAM- 1 levels are commonly associated with poor prognosis (Tang and Hom. 1994b).

PECAM- IlCD3 1 has been studied widely due to its diverse roles in different

circurnstances. PECAM- 1 has six Ig domains, a transmembrane domain and a

cytoplasmic tail (Newman et al., 1990, Simmons et al.. 1990). The cytoplasmic tail of

PECAM- 1 in mouse is encoded by eight exons. which can be altematively spliced or

modified to yield PECAM-I proteins with changed adhesive capacities (Baldwin et al.,

1994). Processing of PECAM- 1 aiso leads to the synthesis of soluble forms of this

molecule (Goldberger et al., 1994). PECAM- 1 is capable of both homophilic and

heterophilic adhesion (Newton et al., 1997). Heterophilic interaction with integnn a&

has been discovered for PECAM-1, although the nature of their interaction is still

unclear. PECAM- 1 has ken shown to play a critical role for most blood cells dunng

inflammation (Carlos and Harlan, 1994). The PECAM- I mediated adhesion between

leukocytes and endothelial cells probably involves homophilic PECAM- I interaction and

heterophilic interaction with a,& (Piaii et al., 1995). PECAM- I concentrated in

endothelial cell-ce11 contacts has been suggested to provide a haptotactic gradient to

guide leukocytes through the endothelium (Dunon et al.. 1996). There is evidence

suggesting that PECAM- I is phosphorylated at specific tyrosine residues, which may

regulate its association with the cytoskeleton (Uan et al., 2000). Furthemore, PECAM- 1

may bind to p-catenin and y-catenin, showing a broader involvement of this molecule in

ce11 adhesion (Dan et ai., 2000).

CD44 is another important cell adhesion molecule that plays important roles in

both physiological and pathologicd process. CD44 is a transmembrane ce11 adhesion

molecule of the hyaladhenn family, a farnily which recognizes hyaluronic acid (Sherman

et al., 1994). CD44 may also interact with other extracellular matrix proteins such as

collagen and fibronectin. Similar to PECAM- 1, many different isoforms of CD44 are

found as a result of alternative splicing and post-translational modification. CD44 has

been shown to be involved in leukocyte diapedesis and lymphocyte activation (Johnson et

al., 2000). It is reported that CD44 cm be used by tumor cells to adhere to endothelium

prior to extravasation (Price et al., 1996). Introduction of CD44 variants into non-

metastasizing tumor cells resultes in metastatic ability of these cells. In melanoma cells,

CD44 is required for migration on type N collagen and for invasion of the basement

membrane (Knutson et al., 1996).

2.4, Cadherins

Cadherins are ~a"-dependent ce11 adhesion molecules mediating cell-ce11

adhesion by homophilic interactions (Takeichi, 1990; Magee and Buxton, 199 1).

Cadherins mediate the formation of adherens junctions, which constinite an important

form of intercellular adhesion structures in al1 solid tissues of the body (Drubin and

Nelson, 1996). In epithelium, for example, continued expression and hnctional activity

of E-cadherin are required for cells to remain tightly associated (Gumbiner, 1996). It has

been well characterized that the loss of expression of E-cadherin is a major Factor in the

development of cancer from epithelial cells (Hirohashi, 1998). in addition to the ability of

mediating the formation of intercellular junctions, cadherins are capable of inducing ce11

polarity (Nathke et al., 1993). For exarnple, E-cadherin has been shown to regulate the

distribution of ~a*lK+-ATPase, potassium channel. and the epithelial growth factor

receptor within the apical surface of epithelial cells ( M m et al., 1993). Since my thesis

project is focused on the role of cadherins in metastasis, the structure and function of

cadhenns will be reviewed in greater detail below.

2.4.1 Structure and function of cuàherins

Cadherins belong to a large, genetically-related gene family (Pouliot, 1992;

Suzuki, 1996). Al1 cadherins are type-1 trammembrane glycoproteins, except for T-

cadherin, which lacks a cytoplasmic tail and is associated with the ce11 membrane via a

glycosylphosphatidylinositol (GPI) anchor (Ranscht and Dours-Zimmermann, 199 1 ).

Cadherins are conventionally categorized into two major groups: classical

cadherins and proto-cadhenns. Classical cadherins. including E-P-M-/Rcadherin,

desmosornal cadherins, T-cadherin, Drosophila fat, the proto-oncogene ret, and others,

contain five extracellular (EC) cadherin domains (usuaily denoted as EC 1-5, starting

from the N terminus) and are distinguished fiom other members of the superfamily by the

O Extracellular dornain

Nucleus

Fig. 2. Schematic diagram of the composition of a classical cadherin complex

and the involvement of p-catenin in signal transduction.

presence of a conserved cytoplasmic tail that associates with cytoplasmic proteins,

(Takeichi, 1995; Yap et ai., 1997) (Fig. 2). The HAV motif, responsible for homophilic

binding activity, is localized in EC I of classical cadherins (Blaschuk et al., 1990). On the

cytoplasmic side, catenins serve as the link between cadherin and the actin cytoskeleton

(Ozawa et al., 1990; Rimm et al., 1995). Importantly, adhesion by classical cadherins is

mediated by the cadherin-catenin complex as a whole (Aberle et al., 1996). Although the

ectodomain alone possesses homophilic-binding properties (Brieher et al.. 1996), stable

cell-ce11 adhesion requires the cadherin cytoplasmic tail and associated proteins

(Nagafuchi and Takeichi, 1988; Ozawa et al., 1990). Recent studies have focused on the

contribution of each of these components to cadherin-based adhesion.

The mechanisms of the homophilic binding activity of classical cadherins have

been studied extensively in tems of their molecula. structure in recent years (Tamura et

al., 1998; Shan et ai., 1999; Leckband and Sivasankar, 2000). It has been demonstrated

that classical cadherins dimerize on the ce11 surface and that cis dirnerization is necessary

for adhesion (Alattia et al.. 1997). The structural data coilected from crystals of the EC 1

and EC2 of E-cadherin and N-cadherin indicate that the extracellular repeat adopts a

seven-stranded p-barre1 structure, which is common among ce11 adhesion molecules

(Overduin et al., 1995: Shapiro et al., 1995: Nagar et ai., 1996; Tarnura et al., 1998).

Three ~ a " ions are bound at the linker region between these two EC domains (Overduin

et al., 1995; Nagar et al.. 1996).

The crystal structure from the flrst EC domain of Ncadherin suggests parallel

interaction between two neighboring cadhenn molecules of the sarne cell, i.e., cis

interaction (Shapiro et ai., 1995). Shapiro et al. observed that the cis interaction is

facilitated by the exchange of N-terminal P strands of the fint EC domain of N-cadherin,

involving the intercalation of Trp-2 residue in the conserved hydrophobic pocket of the

partner molecule. The Ala residue of the HAV homophilic binding motif contributes to

the formation of this pocket. However, different models of cis interaction have been

suggested based on the crystai structure of the first two EC domains of E-cadherin and N-

cadherin. An overall similar cis dimer has been observed in the crystal structure of the

first two EC domains of E-cadherin (Nagar et al., 1996). However, the inter-molecule

exchange of the Trp-2 residue was not observed. Trp-2 is disordered in this structure.

Three calcium-binding sites have been defined to be at the region connecting EC 1 and

EC2. The cis interaction is proposed to be on the calcium binding face, involving residues

in the linker region. A similar mode1 based on the data from the fint two EC domains of

N-cadherin has been proposed by Tamura et al. (1998) . Here Trp-2 is also disordered but

the cis interaction is different from that in Nagar's model. The interface is close to the

Iinker region as well, but smaller and primarily water-mediated, involving only two

hydrogen bonds between the two neighboring molecules. A new structure of the first two

EC dornain of E-cadherin has been resolved recently by Pertz et al. ( 1999). The Trp-7

residue was clearly visible in this structure and found to be docked in the hydrophobic

cavity formed by residues of its own peptide, instead of the partner molecule as observed

in Shapiro's model.

Compared to the cis interaction, the trans interaction between two EC domains of

cadherins on apposing cells is poorly defined. The conserved HAV motif in EC 1 of

classical cadherins has been identified as the major homophilic binding site (Blaschuk et

ai., 1990). The HAV motif is localized in the tram interaction interface in Shapiro's

mode1 (Shapiro et ai., 1995). However, no such interface has been observed in other

models. In Pertz's rnodel, the Trp-2 is important for trans interaction (Pertz et al., 1999).

Trp-2 docks into a hydrophobic cavity of its own molecule and mutation of Trp-2 to

alanine or Ah80 to isoleucine (which blocks the pocket for tryptophan docking) leads to

a complete loss of tram-interactions without affecting cis-dimenzation.

2.4.2 Cytoskeletal interaction of cadherins

The cytoplasmic domains are the most conserved regions of the classical

cadherins. These domains are associated with intracellular proteins, such as

p-catenin, y-catenin, and p 120"" (Ozawa et al., 1990; Aberle et al., 19%). The catenins

are thought to mediate the interaction between cadhenns and the cytoskeleton.

There is a conserved binding domain for P- and y- catenin in the C-terminus of

classical cadherins. Numerous studies have demonstrated the critical role of the

cytoplasmic tail, particularly the catenin-binding site for cadherin-mediated cell-ceil

adhesion. Deletion of the cadhenn cytoplasmic tail or the catenin-binding site alone

abolishes stable cadherin-mediated aggregation of cultured cells (Nagafuchi and

Takeichi, 1988). p-catenin binds directly to the cadherin cytoplasmic tail and serves as a

linker to a-catenin (Hulsken et al., 1994), which interacts directly with cytoskeleton

(Aberle et al., 1994; Hulsken et al., 1994). p-catenin is occasionally substituted by y-

catenin (aiso called plakoglobin) in the cadherin-catenin complex (Hulsken et ai., 1994).

y-catenin is a major component of desmosomes, where it binds to desmosomal cadhenns

(Witcher et al., 1996). Both p-catenin and y-catenin show significant homology to the

Drosophila segment polarity gene amadillo (Butz et al., 1992). The central region of

these homologous proteins contains a 42 amino acid motif repeated 12-1 3 times. These

repeats were originally identified in armadillo, and are termed armadillo repeats. It has

been hypothesized that the arrnadillo repeats function as stnictural domains providing a

scaffold for protein-protein interactions (Aberle et al., 1996). p 12OC". another major

component of the cadherin complex, is also a member of the amadillo farnily (Reynolds

et al., 1994). The role of p 120'" will be discussed later in this session. p-catenin and y-

catenin bind with a-catenin. which, in turn. may bind directly to actin (Rudiger, 1998).

Three domains in a-catenin contain 30% sequence identity with vinculin, implicating its

function as a linker between the cadherin-catenin complex and the cytoskeleton. a-

catenin is also necessary for cadherin adhesion function (Hirano et al., 1992). In some

cases, mutations in a-catenin lead to the loss of cadhenn function, even when cadherin is

expressed normally (Buliions et ai., 1997).

Given their roles in tissue rnorphology, cadhenn-mediated cell-ce11 contacts are

able to provide irnmediate local signais that influence ce11 shape and behavior. It has been

demonstrated that, in addition to the role as a linker, b a t e n i n is also involved in the

signal transduction process (Barth et al., 1997; Gumbiner, 1997; Ben-Ze'ev and Geiger,

1998) (see Fig. 2). p-catenin and armadillo in Drosophila and Xenopcu have been shown

to participate in embryonic induction events as intracellular components of the Wnt

signaiing pathways (Huber et al., 1996a). In cultured rnammalian cells, transient nuclear

localization of ptatenin has been discovered, suggesting a role of p-catenin in

transcriptional reguiation (Huber et al., 1996b; Kuhl and Wedlich, 1997; Fagotto et al..

1998). Another cytoplasrnic protein, the colon carcinoma tumor suppressor gene

adenornatous polyposis coii (APC) protein, has k e n demonstrated to be a crucial

component in the p-catenin signaiing pathway (Rubinfeld et ai., 1993). Sequence

analysis shows that APC is also distantly related to the armadillo family. pcatenin c m

form a complex with APC upon phosphorylation, and thus APC functions as a sequester

of p-catenin (Rubinfeld et ai., 1996). The P-catenin-APC complex is then targeted for

degradation. Mutation in APC cm result in the loss of ability to bind p-catenin, which in

tum Ieading to elevated levels of p-catenin in cpoplasm (Polakis, 1995). Increased

cytoplasmic levels of p-catenin can induce the nuclear translocation and the activation of

Wnr genes by the complex of p-catenin and memben of the lymphoid enhancer binding

factor (LEF)/T-cell-specific factor (TCF) farnily (Korinek et ai., 1997). Furthemore,

mutations of p-catenin of sirnilar consequence are also found in melanoma (Rubinfeld et

al., 1997). These mutations may alter the bind of p-catenin to APC and the regulation of

protein degradation, resulting in increased levels of cytosolic p-catenin and enhanced

formation of the P-catenin-LEF-1 complex. Several target genes that may be activated by

this complex have been identified so far, such as the transcription factor Tcf4 (Roose and

Clevers, 1999; Roose et al., 1999).

There is increasing evidence suggesting that the cytoplasmic component, p 126'",

also plays important roles in the regulation of the function and organization of cadherin

complexes (Provost and Rirnm, 1999). p 1 2OCm, aiso termed ka ten in , binds to the

membrane-proximal region of the cytoplasmic tail of cadherins. p 120'" is a member of

annadillo gene farnily and is originaily identified as a subsuate of src-kinase (Reynolds

et al.. 1992). Different groups have proposed conflicting models on the role of p 1 20Ctn in

cadherin complexes. Ozawa and Kernler ( 1998) showed that deletion of 144 or 15 1

amino acid residues from the C-terminal region of E-cadhenn, which eliminated the

p l 20cm binding site, resulted in adhesion activity as strong as that of w ild type E-cadherin.

Therefore. it is proposed that p 120Cm prevents dimerization and negatively regulates

adhesion activity of E-cadhenn. However, totally different results are reported based on

the data obtained using Xenopiis C-cadherin lacking the juxtamembrane region (Yap et

al., 1998). Mutant C-cadherin molecules lacking either the complete cytoplasmic tail or

just the juxtamembrane region do not cluster whereas the juxtamembrane region itself is

sufficient to induce clustering when hsed to a heterologous membrane-anchored protein.

These observations lead to a mode1 that the juxtarnembrane region of the cadherin

cytoplasmic tail functions as an active region that supports cadherin clustering and

enhances adhesive strength. Recently, observations make by Takeichi's group suggest the

possibility to reconcile these controversid results (Aono et al., 1999). They have reported

that cadherins function normally if they do not bind to p l2OCm. However. p 120"" acts as

an inhibitory regulator of cadherin function in colon carcinoma cells in a

hyperphosphorylated form. Therefore, it has been hypothesized that the phosphorylation

state of p 120'" cm be modulated and that depending upon the phosphorylation state,

p12OCm c m exert different effects, providing a new mechanism for the functional

regulation of cadherins.

2.4.3. The role ofcadhenn complex in the development of cancer

It has been recognized for many years that the transition from a normal ce11 to a

tumorigenic ce11 includes changes in intercellular adhesion (Brodt, 199 1; Fawcett and

Harris, 1992). One important feature of cancer is the alteration of the way that cancerous

cells interact with the surrounding tissues, such as the loss of contact inhibition and easy

detachment from the pnmary foci. Considerable evidence has suggested that cadherins

play important roles in many different cancers. For example, E-cadhenn function is

frequently inactivated during the development of human carcinomas, including those of

the breast, colon. prostate. stornach, liver. esophagus, skin, kidney, and lung (Birchmeier

and Behrens. 1994: Bracke et al., 1996). Abrogation of E-cadherin function may occur by

several mechanisms. but it frequently involves deletion or mutation of the E-cadherin

gene (Birchmeier and Behrens. 1994; Bracke et al.. 1996). Loss of E-cadhenn in

epithelial cells leads to improper organization of epithelial cells and much higher

tendency of malignancy. Besides the mutations in the E-cadherin gene, changes in the

expression of proteins that are components of the cadherin complex have been found to

impair E-cadhenn-mediated cell-ce11 adhesion, such as down-regulation or mutation of a-

catenin or B-catenin (Semb and Christofori, 1998). Mutations of the B-catenin gene are

frequently found in colon cancers (Sparks et al., 1998). In various cell-culture systems,

the re-establishment of a hnctionai cdhendcatenin complex causes invasive tumor ce11

lines to revert to a benign, epithelial cellular phenotype (Frixen et al., 199 1 ; Birchmeier

and Behrens, 1994; Bracke et ai., 1996). Thus, E-cadherin is considered to be an

important tumor suppressor gene.

2.4.4. Glycosylation of cadherins

Glycosylation is a cornmon post-translational modification for ce11 surface

proteins. Aberrant glycosylation in tumor cells has been implicated as an essential

element in defining the stage, direction. and fate of tumor progression (Demis et al.,

1999). Numerous clinical and pathological studies have revealed a correlation between

aberrant gl ycos ylation status of primary tumors and their invasive/metastatic potential.

For example, expression of B-N-acetylglucosaminyltransferase-Tm (GlcNAc-TU1 or

GnT-III) and TV (GlcNAc-TV or GnT-V) in the liver is enhanced during

hepatocarcinogenesis. although they are not expressed in the normal liver (Miyoshi et al..

1993). In metastatic B 16-hm murine melanoma celIs, however, introduction of the GnT-

[II gene was reported to suppress metastasis (Yoshimura et al., 1995).

The glycosylation of cadherins has not been thoroughly studied. yet there is

evidence to suggest that the glycosylation of E-cadhenn, which is mediated by GnT-UI,

is able to regulate the adhesion activity of cancerous cells (Yoshimura et al.. 1996). E-

cadherin glycosylated by ectopically expressed GnT-JIi exhibits delayed tumover and

decreased release from the cell surface compared with native E-cadherin, resulting in

elevated expression at the cell-ce11 border of GnT-III transfectants. Thus it has been

postulated that the glycosylation may influence the tumover of E-cadherin molecules on

ce11 surface, as well as the adhesive strength (Yoshimura et al., 1996).

3. Role of Gap Junctions in Interaction between Cancer CeIl and the Endothelium

Gap junctions are intercellular channels hinctioning as an exchange pathway of

small molecules and signals between adjacent cells (Goodenough et al., 1996). In

vertebrates, the structural proteins of gap junctions are encoded by a multi-gene family,

the connexins. Connexins have four transmembrane domains and fourteen mouse

connexin genes have been cloned so far. Six connexin molecules fom a structure named

hemi-connexon. Two hemi-connexons kom two neighboring cells form a gap junction

with an aqueous intercellular channel. The pore size of the connexon channel allows the

passage of only small molecules under 1200 Da, such as water, amino acids, second

messengers and small metabolites (Simpson et al., 1977). Gap junctions play important

roles in signal transduction and ce11 coupling. For example, the synchronized contraction

of cardiac and smooth muscle cells is mediated by gap junction. The composition of gap

junctions can be homogenous or heterogeneous. In most cases. gap junctions consist of

only one type of connexin. Sometirnes, more than one type of connexin cm also form a

functional connexon.

It has been demonstrated that gap junctions are involved in tumor progression. A

large amount of evidence suggests that in many cases cancer cells lose the ability to

communicate via gap junctions (Trosko and Ruch. 1998). Therefore. cancers c m be

considered as a 'disease of homeostasis'. The abnonndities of gap junctions in cancers

are not only malfunctioning, but cancer cells can also establish gap junctiond

communication with wrong types of cells. The coupling with inappropriate cells may

promote cancer ce11 proliferation in organs and rnicroenviroments totally different from

their prirnary sites. It has been suggested that tumor cells can fom gap junctions with

endotheliai cells (el-Sabban and Pauli, 1994). The main connexins expressed in

endothelid cells are connexin43,37 and 40 (Te10 et al., 1997). Melanoma cells rnay form

connexin43 mediated gap junctions with endothelial cells in an adhesion-dependent

fashion (el-Sabban and Pauli, 1994). Moreover, dye transfer can be shown to occur upon

attachent of labeled tumor cells onto endothelial cells, and higher levels of connexin43

are associated with increased metastatic potential of melanoma cells (el-Sabban and

Pauli, 199 1).

4. Endothelial Cell-CeII Contact

The endothelium of blood vessels constitutes a physical barrier to cells in the

circulatory system. For cells to escape the circulatory system. such as lymphocytes

reacting to immune responses or metastatic cancer cells, they have to penetrate through

the interendothelid ce11 junctions to migrate into the underlying tissue (Fig. 3).

Endothelial cells express two major cadhenns: VE-cadherin and N-cadherin

(Dejana, 1997). VE-cadherin is an atypical cadherin predominantly expressed by

endothelial cells and plays very important roles in the development and integrity of

endotheliurn. VE-cadherin has several features of classical cadherins, such as five

extracellular repeats and conserved cytoplasrnic tail. However. it lacks some other

features including the absence of the HAV motif. In mouse embryoid bodies that contain

a mutated VE-cadherin gene, endotheliai cells remain dispersed and fail to organize into a

vessel-like pattern, even though they express a large range of other endothelial markers

(Carmeliet et al., 1999). While VE-cadhenn is localized at endotheliai cell-ce11 contact

region, Ncadherin is only difisely disuibuted on the ce11 surface of endothelial cells

(Salomon et al., 1992). Recent studies suggest that VE-cadherin may compete for the

cell-cell contact regions with N-cadherin being excluded from the endothelial junction

(Navmo et al., 1998). The role of N-cadherin in the mature endothelium is unclear. It is

possible that N-cadherin may play a role in the anchorage of endothelial cells to other N-

cadherinexpressing cells, such as smooth muscle cells, astrocytes or pencytes (Dejana,

1997).

Tight

Adherens junction- -

Fig. 3. Schematic representation of the molecular complexes localized at

interendotheiial junctions.

PECAM- 1KD3 1 is the major Ig-CAM present on endothelial junctions and is

capable of mediating both homophilic and heterophilic interactions (Newman, 1997). In

endothelium, PECAiM- 1 distributes evenly dong the entire endothelial contact region

(Ayalon et al.. 1994). PECAM-I is known to be involved in regulating the diapedesis

process of lymphocytes (Muller et al.. 1993). The attachment of leukocytes leads to the

dissolution of adherens junctions. whereas PECAM- 1 remains concentrated in endothelial

junctions during leukocyte transmigration. Pretreatment of neutrophils and monocytes

wi th anti-PEC AM- 1 antibodies blocks the transendothelial migration of these leukoc ytes

(Newman 1995, 1996).

Another major adhesive complex in the endothelium junction is the tight junction.

Tight junction is the most important regulator of paracellular permeability in well-

established epithelium (Anderson and Van Itallie, 1995; Mitic and Anderson, 1998:

Stevenson and Keon. 1998). Tight junctions are dso responsible for regulating the

pemeability of endothelia and demarcate the apical and basolateral membranes of

endothelid cells. In the lung microvascular endothelial cells, tight junctions are not as

well developed as those endothelia that strictly control exchanges between blood and

tissues (typically at the blood-brain bmier and in the large arteries) (Lampugnani and

Dejana 1997). Tight junctions are constituted by the membrane protein occludin and

cytoplasmic proteins, such as 20-1,ZO-2 and 20-3 (Mitic and Anderson, L998). The

cytoplasmic proteins connect the intracellular tail of occludin to the cytoskeleton.

5. Invasion of Target Organs by Cancer Cells

It has long been recognized that the dissemination of cancer cells in the body

follows discrete patterns. For example, small ce11 carcinomas preferentially metastasize to

brain while prostatic carcinoma metastases are commonly found in bone (McKinnell et

al. 1998). If tumor cells escape from the vasculature at random sites. there will be no

pattern of specific target organs associated with a particular cancer. About a hundred

years ago, &ter observations of over seven hundred patients who died from breast cancer.

Paget found that liver is the major organ of metastasis. Thus he proposed the "seed and

soil" theory and hypothesized that cancer cells are like seeds that only grow in soil where

the growth is favored (Paget. 1889, republished in 1989). The microenvironment is

cnticai for the formation of secondary metastasis. For each particular cancer, there rnight

be some vasculature optimal for their extravasation or some organs that are favorable for

their proliferation. Recent studies suggest that interaction of ce11 adhesion molecules

expressed by both cancer cells and endothelial cells play an important role in the arrest

process. Specificity at the site of anest has been idrntified as one important factor

contributing to the organ-specific metastatic patterns of many cancers (Brodt, 1989:

Scherbarth and Orr, 1997). Possibly. a particular cancer may interact preferentially with

some endothelia. Furthermore, this idea is supported by the observation that vascular

beds in different organs show antigenic differences. For example, lung endothelial

adhesion molecule-1 (Lu-ECAM- 1), an Ig-CAM, is expressed only by endothelid cells

of the lung. A correlation between the expression of Lu-ECAM- 1 and the location of

melanoma metastasis has been reported. Lu-ECAM-1 has been shown to participate in

the adhesion of melanoma cells to lung endothelium in vivo (Zhu and Pauli, 1993).

An alternative model was proposed to interpret the organ preference of cancers

(Fidler and Talrnadge, 1984). It is a cornmon concept that circulating cancer cells need to

be arrested before they can undergo extravasation from the blood Stream. Morphologicai

evidence indicates that only single cancer cells can enter and be arrested in the capillaries

(Starkey et al., 1984; Weiss et al.. 1992). In this model, the organ preference is the result

of entrapment of cancer cells in the first capillary bed encountered. Therefore. the

location of a metastasis may be related to blood-borne cells being trapped with higher

frequency in some organs rather than organ specific.

In fact. the pattern of organ involvement in metastasis probably depends on both

the mechanical sieving effect of capillary beds and the presence of specific environment

in certain organs that favor the adhesion, extravasation and proliferation of tumor cells.

6. Melanoma Cells as A Mode1 System

Melanoma has been one of the fastest rising malignancies in the last 4 decades.

with the incidence increasing from less than 3 per 100,000 individuais to more than 12

today (Parker et al., 1997). By the year 2000, it is estimated that 1 in 70 people in the

United States is expected to develop melanoma over hdher lifetirne. Melanoma is

particularl y notorious for its potentiai to metastasize and for poor vitality after metastasis.

Melanoma cells are being used extensively in many in vitro and in vivo studies.

Melanomas have been categocized in tems of their stages of tumor progression. Many

melanoma ce11 lines have been isolated and established from each of these stages.

Melanoma cells originate from melanocytes. Based on the clinical and

pathological features, the progression of melanoma is divided into five steps: cornmon

acquired nevus. dysplastic nevus, radial growth phase (RGP) p n m w melanoma,

tumorigenic vertical growth phase (VGP) pnmary melanoma. and metastatic melanoma

(Meier et al.. 1998). Melanocytes in the normal skin reside in the basement membrane,

which separates that epidermis from dermis. They adhere to keratinocytes through E-

cadhenn, which allows the formation of gap junction-mediated channels between the

cells (Hsu et al.. 2000). The keratinocyte controls the growth and phenotype of the

melanocyte. The melanocyte c m escape this control when E-cadherin expression is shut

off (Hsu et al.. 1996). When successful, the melanocyte cm proliferate to form a cell

cluster. i.e., a nevus, which remains an accumulation of stmcturally normal cells.

Genetic changes are anticipated when dysplastic nevi develop. but the nature of

these changes is currently unknown (Morita et al., 1998). In some cases. the ceil cycle

checkpoint pathways (including p53 and myc) are found to be involved in the

development of dysplastic nevi (Chenevix-Trench et al., 1990). The transition from RGP

to VGP melanoma is a biologicaily and clinically critical step. accompanying additional

genetic abnormalities (Satyarnoorthy et al.. 1997). Unlike RGP melanomas. VGP cells

are metastasis-competent and are easily adapted to growth in culture (Guerry et al..

1993). In addition, VGP cells are less dependent on exogenous growth factors and have

growth charactenstics sirnilar to metastatic cells, such as anchorage-independent growth

in soft agar and tumorigenesis in immunodeficient mice. No major additional genetic

changes may be required for further progression to metastatic melanoma since most VGP

melanomas can be readily adapted to a metastatic phenoiype through selection in growth

factor-free medium or induction of invasion through artificial basement membranes (Kath

et al., 1991).

The preferential organs for metastases of melanorna are lung. brain and liver. Our

laboratory h a , therefore, chosen to study the mechanisms of interaction between

melanoma and human lung microvascular endothelial cells. An in vitro mode1 system has

been developed in our laboratory for this purpose (Fig. 4) (Sandig et ai., 1997).

Endothelial cells are cultured on round 12-mm glass coverslips coated with Matrigel. The

Matrigel provides an appropnate layer of extracellular matrix for endothelial cells and

allow them to differentiate and form a thin monolayer. Melanoma cells labeled with DiI,

or other fluorescence dyes are then added on the monolayer, and the CO-cultures are

incubated for different time intervals (1-5 h) before fixation and immunostaining (Fig. 4).

Five stages of transmigration of melanoma cells are distinguished based on their

morphology (Voura et al., 1998a). Stage I represents the initial attachrnent of melanoma

cells to the endotheliai monolayer. Not many changes occur at this stage. Stages II and El

are the rniddle stages of transmigration. In stage II, numerous membrane blebs c m be

observed in the region where melanoma cells corne into contact with endothelial cells. In

stage III, pseudopodia of melanoma cells start to penetrate into endothelial ce11 junctions.

Endothelial cell-ce11 contacts undemeath the Nmor ceil start to dissociate. Stage IV and V

are the late stages of transmigration. Melanoma cells in stage N become intercalated

among endothelial cells and the ventral surface begins to spread on the underlying

Matrigel. Endotheliai ceils begin to spread on the top of tumor ce11 body and reseal the

gap caused by the melanorna cell. In stage V, melanoma cells complete penetration and

spread on the Matrigel, adopting a fibroblastic morphology. Endothelial cell-ce11 contacts

reestablish over the tumor cells. In this in vitro transendotheliai migration assay, tumor

Coat coverslip with Matrigel

Add endothelial cells and dlow to settle for 3 h

Stimulate endothelial cells with tumor necrosis factor a

Add melanorna cells

Stages of Transendothelial Migration

Figure 4. The in vitro mode1 assay of transendothelial migration of

melanoma cells.

cells adopting the morphology of stages N and V are scored as cells that have completed

transmigration.

7. Previous Work on CeU Adhesion Molecules in Our Laboratory

Different models have been proposed to describe the extravasation process when

cancer cells transmigrate from the apical surface of the endothelium to the basal-lateral

side. Scanning electron microscopy has suggested that endothelial ceils retract upon

interaction of cancer cells. Then the cancer cells migrate through the gap, confemng only

transient direct contacts with endothelial ceIls (Lewalle et al., 1997). However, active

involvement of both cancer cells and endothelial cells has been suggested in Our in vitro

studies (Voura et al.. 1998b). The penetntion of melanoma cells is initiated by the

appearance of numerous membrane blebs protruding from the basolateral surface of

rnelanoma cells. At the same time, abundant actin filaments appear from the underlying

endothelial cells at the regions of contact. These interactions lead to the reorganization of

the cytoskeieton and the redistribution of VE-cadherin and PECAM- I in endothelial

cells, resulting in the local dissolution of the endothelial junction undemeath the

melanoma cell. In our in vitro system, no significant arnount of occludin. the key

molecule of tight junctions, was observed between endothelial cells. Direct contact

between melanoma cells and endothelial cells is maintained throughout the penetration

process, suggesting adhesive interaction between these iwo ce11 types.

Previous members of our laboratory have also investigated the role of cadherins in

melanoma transendothelial migration. In the early stages of diapedesis, VE-cadherin

redistributes away from the endothelial contacts located undemeath the melanoma cell.

hunofluorescence staining using a pan-cadherin antibody, which recognizes the

conserved cytoplasmic tail of classical cadherins, shows the presence of classical

cadherins in the heterotypic contacts between endothelial cells and WM239 melanoma

cells (Sandig et al.. 1997). However, the identity of this classical cadherin rernains to be

defined. Upon the completion of transmigration by melanoma cells, VE-cadherin-rich

cell-ce11 contacts re-establish between endothelial cells spreading above the melanoma

cell. Furthermore, addition of anti-N-cadherin antibodies to the assay leads to partial

inhibition of the transendothelial migration of melanoma cells. suggesting a role for N-

cadherin in this process (Sandig et al., 1997).

The role of PECAM-I (CD3 1) has also been examined by Voura ( 1999). As

mentioned previously, PECAM- 1 is an important Ig-CAM present on endothelial contact

regions. PECAM-1 has been proved to regulate the diapedesis process of leukocytes.

Transendotheliai migration of leukocytes can be blocked by anti-PECAM- 1 antibodies

(Muller et al.. 1993). However. the same blocking mAbs against PECAM-1 are unable to

inhibit the transendothelial migration of melanoma cells. Furthermore. adhesion

complexes formed by PECAM- 1 undergo localized dissolution upon contact with

melanoma cells, sirnilar to previous observations of VE-cadherin (Lewalle et al., 1997:

Sandig et ai., 1997). Thus, melanoma cells adopt a different strategy to migrate through

the endothelium.

Another cell adhesion molecule that we have studied is the a& integnn.

Melanoma cells express increased levels of during tumor progression (Meier et al.,

1998). Immunofluorescence labeling studies show that integrin is present in the

heterotypic contacts region between endothelial cells and melanoma cells. The

transmigration of meianoma cells is inhibited by the addition of antibodies against or

a& and cyclic RGD peptide (Ruoslahti. 1996; Voura. 1999). Furthemore, a&-

deficient M2 1 melanoma cells migrate poorly compared to their a$3-positive

counterparts. The interaction between and its ligand LI has been proposed to

mediate melanoma transendothelial migration (Voura, 1999).

8. Hypothesis, Rationale and Objectives of the Thesis

The overall purpose of this study is to elucidate the molecular mechanisms

involved in the extravasation of tumor cells. The main focus of my thesis is to test the

hypothesis that N-cadherin is involved in the transendothelial migration of melanoma

cells and to investigate its role in this process.

As introduced in the previous section, previous data in our laboratos, suggest the

involvement of classical cadherins in the interaction between WMS39 melanoma cells

and endothelial cells. inhibition assay using anti N-cadherin rnAb GC4 shows that

transendothelial migration of WM239 cells is partially inhibited, suggesting the

involvement of N-cadherin. However. immunolabeling of N-cadherin shows that N-

cadherin is only evident in the leading edge of the re-closing endotheliai cells on top of

melanoma ce11 (Sandig et al., 1997). My initial objective was to identify the classical

cadhenn or other ce11 adhesion structures present in the heterotypic contact region

between tumor ce11 and endothelial cells and study their implication in tumor metastasis.

Immunofiuorescence labeling studies and Western blot analysis were carried out

using an anti N-cadherin mAb with high affïnity to human N-cadhenn. Direct

involvement and upregulation of N-cadherin during transendothelial migration of

WM239 cells were demonstrated. The antisense oligonucleotide approach was adopted to

reduce the levels of N-cadherin expression in WM239 ceils. Greater than 80% reduction

in N-cadherin levels was achived in several ce11 clones transfected with the N-cadhenn

antisense vector. The reduction of N-cadherin resulted in partial inhibition of melanoma

transendothelial migration. Changes in the proteins associated with N-cadherin, such as

p-catenin and actin, were also exarnined. Another novel discovery is that WM35 cells

were unable to form N-cadhenn-mediated contacts with endothelial cells and expressed

an N-cadhenn isoforrn that was abnormally glycosylated. in addition to the studies on N-

cadhenn, expenments were conducted to examine the role of gap junctions in melanoma

transendothelial migration. Gap junctions were observed in the heterotypic contacts

between melanoma cells and endothelial cells and the melanoma transendothelial

migration can be partially inhibited with the treatment of the gap junctional

communication inhibitor, 1 -heptanol.

In summary, these studies have shown the important function of N-cadhenn in the

transendothelial migration of melanoma cells. There is increasing evidence that N-

cadherin may play a role in tumor progress, migration and metastasis. Data presented in

this thesis provide new evidence in support of the biological significance of N-cadherin

in cancer metastasis. Future work to dissect the rnechanism of the establishment of N-

cadherin-mediated contacts between endothelial cells and cancer cells should lead to a

better understanding of the exwvasation process of cancer cells.

Chapter 2: Muterials and Methods

Antibodies

Rabbit anti-connexin43 and mouse anti-connexin43 antibodies were purchased

from Chernicon International (Temecula, CA). The mouse anti-actin mAb was purchased

from Boehringer Mannheim (Mannheim, Germany). Mouse anti-human NIEIPNE-

cadhenn, mouse anti-human p-catenin and mouse anti-human p 120C'" mAbs were

purchased from Transduction Laboratory (Lexington, KY) for irnmunofluorescence

staining and Western blot analysis. Rabbit anti-human N-cadherin was purchased from

R&D Systems (Minneapolis, MN) for immunoprecipitation studies. The FITC- and Texas

Red-conjugated goat anti-rnouse and goat anti-rabbit antibodies were purchased from

Molecular Probes (Eugene. OR).

Cell Lines and Culture Conditions

Human lung microvascular endothelial cells (HMVEC) were purchased from

Clonetics (San Diego. CA) and cultured in endothelial growth medium (EGM-2 MV)

(Clonetics) supplemented with penicillin-streptomycin ( IOUIrnl each) (Gibco-BRL,

Burlington, ONT) in a humidified atmosphere containing 5% CO?. The melanoma ce11

lines, WM239 and WM35, were obtained from Dr. Meehard Herlyn (Wistar Institute,

Philadelphia, Pennsylvania) and routinely cultured in RPMI 1640 medium supplemented

with 10% heat inactivated fetal bovine serum (FBS) and penicillin-streptomycin ( 10 U/ml

each). The features of these melanoma ce11 lines are summarised below (Table 1).

Transendothelial Migration Assay

Table 1: Characteristics of melanoma ce11 lines used in experiments.

Round glass coverslips ( 12 mm in diarneter and 0.13-0.17 mm thick) (VWR

Canlab, Mississauga, ON) were coated with Matrigel (at 1 :8 dilution) (Becton/Dickinson.

Bedford, MA). Matrigel was diluted with icesold water and 100 pl was applied to each

coverslip in 24-well plates. The coverslips were air-dned ovemight in a laminar flow

hood at room temperature and then rehydrated in Hanks' buffered saline solution (HBSS)

(Sigma, Oakville, ON). After rehydration, the Matrigel fomed a thin Iayer, which would

optimise endothelial ce11 attachment and the formation of a monolayer. HMVEC cells

(1.5 x lo5 cells taken between passages 4 and 8) in a 200 pl drop of endothelial growth

medium were placed on top of the Matrigel-coated coverslips and allowed to settle for 5-

6 h. Covenlips were then transferred to 24-well plate and incubated in 0.5 ml endotheliai

growth medium containing 10 nglml TNFa (GibcolBRL. Burlington, ON). Phase

microscopy showed that confluent monolayers were obtained after 12 h of culture, and

they rarely showed gaps under these conditions.

Ce11 line

WM239

WM35

Stage of isolation

Secondary metastasis

RGP-like phenotype (early

prirnary melanoma):

Tumor growth in nude

mice (mass after 10 weeks)

0.5- 1 .Og

Poor (Bani et al., 1996)

References

(Herlyn et

ai., 1985)

(Herlyn et

d., 1983)

Melanoma cells were removed from culture dishes by incubation in HBSS

containing 4 mM EDTA and subsequently labeled with 2.5 m g h l Di1 (Molecular Probes,

Eugene, OR) for 5 min at 37°C for ce11 counting or 1 pM Celltracker Orange CMTMR

(Molecular Probes, Eugene, OR) for 30 min at 37°C For laser scanning confocal

microscopy. The labeled cells were washed three times in HBSS and the final pellet was

resuspended in endothelial growth medium at a concentration of 2.5 x 106 cells/ml.

Melanoma cells (5 x lo4 cells in 20 jA) were then added on to a HMVEC monolayers and

incubated for different time intervals pnor to fixation and staining.

Fixation and Staining of Cells

Cells on coverslips were fixed using 3.5 % ( w h ) paraformaldehyde in phosphate-

buffered saline (PBS). pH 7.4, ai room temperature for 5 min or 100% methanol

(prechilled to -20°C) on ice for 3 min. The cells were then washed three times in PBS.

The paraformaldehyde fixed cells were subjected to extraction for 5 min in the

cytoskeleton-stabilizing buffer containing 0.1 M 1,4-piperazine-bis(ethanesu1fonic acid)

(Aldrich, Milwaukee, WI), 1.0 mM EGTA, 4% (w/v) PEG8000 and 0.1 % (v/v) Triton X-

100 (pH 6.0) (Opas and Kalnins, 1985). The extraction was followed by another senes of

washes. Before staining, the cells were blocked for 5 min in PBS containing 1% (wfv)

BSA. Cells were labeled with pnmary antibody (l:3ûû) for 45 min and subsequently with

secondary antibody (1:3ûû) for 45 min at room temperature, followed by three washes

with PBS after each labeling step. The coverslips were then mounted for microscopy

using mounting medium that contained 80% glycerol in PBS and 2.5% of the antioxidant,

1,4-diazabic yclo- [2,2,2]-octane (Sigma, Oakville, ON).

Fluorescence Microscopy

Laser scanning confocal rnicroscopy was carried out using a Zeiss Axioven 135

inverted microscope which was equipped with a 6 3 ~ Neofluor objective and the LSCM

4 10 confocal attachment. Quantitative analysis of transmigration of melanoma cells was

carried out using a Wild Leitz Orthoplan microscope equipped with epifluorescence

optics.

Quantification of Transmigration by Melanoma CeIls

Three sets of random fields for a total of 45 fields were scored for each coverslip.

Each set of 15 fields contained 100-250 melanoma cells. Al1 ce11 counts were carried out

using F-actin stained coverslips. with melanoma cells pre-labeled with Di1 for

identification. Melanoma cells were divided into three major categories as described in

Fig. 4. The percentage of transrnigrated melanoma cells was calculated for each fifteen-

field set separately. Experiments were repeated independently 3 times for each condition.

Immunoprecipitation

Cells were washed twice with icetold HBSS containing 2 rnM ca2+ and lysed by

incubation for 30 min on ice in a lysis solution containing 10 mM Tris-acetate (pH 8.0),

150 mM NaCl, 0.5% NP-40,l mM EDTA, 1 mM EGTA, 1 mM PMSF, I mM sodium

oahovanadate, and protease cocktail at 1: 10 dilution, which contains 100 rnM 4-(3-

aminoethyl)benzenesulfonyl fluoride (AEBSF), 1.5 mM pepstatin A, 1.4 mM tram-

epoxysuccinyl-L-leucylamido(4-guanidino)ute (E-64). 4 rnM bestatin. 2.2 rnM

leupeptin, and 0.08 rnM aprotinin (dl from Sigma). Cellular debns was pelleted by

centrifugation at 10,000 rpm for 10 min at 4°C. The supernatant was incubated ovemight

with 2 pg of pnmary antibody at 4°C. Then the supernatant was incubated with LOO mg

protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway. NJ) for 1 h at 4°C. The

protein A-Sepharose was equilibrated in lysis solution before use. The protein A-

Sepharose and protein complexes were separated by centrifugation at 10,000 rpm for 10

min at 4 ° C washed three times in 10 mM Tris-HCl (pH 8.0). 150 mM NaCl and 0.5%

Tween-20. Proteins in these samples were solublized by incubation in Laernmli's buffer

containing 2% ( d v ) SDS, 10% glycerol (v/v), 60 rnM Tris-HCl (pH 6.8) and 0.001 %

(w/v) bromphenol blue and 2% (vfv) P-mercaptoethanol, at 95°C for 10 min. and then

subjected to separation by SDS-PAGE (Laemmli. 1970).

Western Blot

Protein samples from whole ce11 lysates or imrnunoprecipitates were routinely

separated on 8% (w/v) SDS-polyacrylamide gels. Proteins were transferred to

nitrocellulose membrane by electroblotting (Towbin et al., 1979). The membranes were

blocked in PBS containing 5% (w/v) skim milk powder and 0.5% (v/v) Tween-20. and

incubated with primary antibodies ( 1 :2500 for actin mAb and 1 : 1000 for other mAbs)

ovemight at 4'C or 1 hour at room temperature. The membranes were washed and

incubated in honeradis h peroxidase-conj ugated secondary antibodies ( 1 : 1000 dilution)

(Bio-Rad, Hercules, CA) for 1 hour at room temperature. The membranes were washed

again and incubated with the ECL detection reagents (Arnersharn Life Science,

Buckinghamshire, UK) and exposed on BioMax X-ray film (Kodak, Rochester, NY). Al1

antibodies were diluted in the blocking solution and ail washes were canied out in PBS

containing 0.5% (v/v) Tween-20. The X-ray films with proper exposure were scanned

and the pixel values were analysed using NM image (ver1.62). For each protein band,

the pixel value obtained from a blank region of the blot with the same size was subtracted

as bac kground.

Ce11 Transfection Using the pBSpacAp9 Vector

The pBSpacApT expression vector was obtained from Dr. Keith Johnson (University

of Toledo, Toledo, OH). The structure of pBSpacAp', which contains the N-cadherin

antisense sequence, is shown in Fig. 5 (Islam et al., 1996). In brief, an 800 bp fragment

containing the human mU6 gene (Noonberg et al., 1994) with proper promoter and

termination signals was cloned into the pPUR vector (Clontech, Pa10 Alto, CA).

Nucleotides in the 3'-end of the original mu6 gene was replaced by a short 25-bp

antisense sequence spanning the start codon of human N-cadherin. The puromycin

resistance gene serves as the selection marker for transfection in mamrnalian cells.

Transfection of the N-cadherin antisense vector pE3SpacApT was c d e d out using

the mamrnalian transfection kit purchased from Stratagene (La Jolla, CA) (Chen and

Okayama, 1987). Before transfection, the melanoma cells were cultured in a-MEM

media supplemented with 10% heat-inactivated fetai bovine semm (FM) and penicillin-

streptomycin (10 U/mi each) in a 100-mm dish. Circular plasmid DNA (20 pg) purified

poly A+ sv40 b

Fig. 5. Schematic drawing depicting the N-cadhenn antisense plasmid, pBSpacAp9.

by the Midiprep Kit (Qiagen, Valencia, CA), was diluted in 450 p i distilled water and

mixed with solution 2 (2 x PBS consisting of 50 rnM N,N-bis (2-hydroxyethy1)-2-

arninoethanesulfonic acid). The mixture was incubated for 20 min at room temperature

and then added CO the ce11 culture. After 24 h, the medium was removed and the cells

were rinsed twice using HBSS before the addition of fresh melanoma medium. The cells

were incubated for another 24 h. The cells were then replated into three 100-mm dishes

and incubated for an additional 24 h before puromycin selection was applied. The

puromycin was added (at a pre-determined concentration of 0.5 pg/ml) to select for

positive transfectants. After 4 weeks of incubation. colonies were isolated and expanded.

Individual colonies were subjected to Western blot analysis to assess the level of N-

cadherin expression and than used in the transendothelial migration assay.

Treatment of CeIls with Antisewe Oligonucleotides

N-cadherin antisense oligonucleotides were purchased from Biognostik (Gottingen,

Germany). WM239 cells were cultured in 2 mM (recornmended) or 4 rnM antisense

oligonucleotides for 1. 2,4.8,24.48 and 96 h. Meanwhile. WM239 cells were treated

with the control oligonucleotides and FITC-conjugated control oligonucleotides at the

same concentration in parallel to the antisense oligonucleotides. Then cells. incubated in

antisense and control oligonucleotides. were collected and assayed for N-cadherin

expression by Western blot anaiysis. The cells incubated in FITC-conjugated

oligonucleotides were fixed with paraformaldehyde as described and subjected to epi-

fluorescence microscopy.

Chapter 3: Results

Part 1: Analysis of the Role of N-cadherin

N-cadherin is Upregulated during Melanoma Transendothelial Migration

Previous work from Our laboratory has suggested the involvement of a classical

cadherin during the transendothelial migration of melanoma cells in vitro (Sandig et al..

1997). My initial objective was the identification of this classical cadherin. First,

expression of classical cadherins, E-cadherin, P-cadherin and N-cadherin, and the

atypicd cadhenn, VE-cadherin in WM239, WM35 and endothelial cells was exarnined.

Total ce11 proteins of WM239, WM35 and HMVEC cells were separated by SDS-PAGE

and then probed with different antitadherin antibodies (Fig 6A). The results showed that

endothelial cells expressed VE-cadherin and N-cadherin. Both melanoma cell lines

expressed N-cadhenn. but not Psadherin. Expression of E-cadherin WM239 is higher

than that in WM35 cells. Therefore, N-cadherin was a good candidate for further

investigation.

Several mAbs against N-cadherin were screened for their ability to recognize N-

cadherin in both imrnunoblots and fixed cells. The anti-N-cadherin mAb purchased from

Transduction Laboratory proved to be most useful. The immunostaining and Western blot

analysis included in this thesis were carried out using this antibody. Immunostaining

studies revealed that N-cadherin was present on the ce11 surface of WM239 cells and

strong signais were obsewed at cell-ce11 contacts (Fig. 6B). E-cadherin staining was very

weak at the ce11 surface and no concentration of E-cadherin was associated with cell-ceIl

contacts. Instead, Etadherin displayed a punctate staining pattern in the c ytoplasm,

suggesting that E-cadherin might be sequestered in small vesicles in Wh4239 cells. P-

cadherin showed negative staining in WM239 cells.

The level of N-cadherin in CO-cultures of melanoma cells and endotheliai cells was

also examined by Western blot anaiysis. A mixture of WM239 cells and HMVEC was

used as a control for the CO-cultures. Thus. any difference between the CO-cultures and the

mixture of single cultures was due to the CO-culturing of WM239 cells and endothelial

cells (Fig. 7A). Protein blots were probed with N-cadhenn rnAb and quantitative analysis

showed a 40% increase in the level of N-cadhenn by 5 h (Fig. 7B). suggesting that the

interaction of rnelanoma cells and endothelial cells might have stimulated an upregulation

of N-cadherin expression. However, it was not clear whether N-cadherin is upregulated in

endothelial celIs or rnelanoma cells or both.

Direct Invohement of N-cadherin in the Interaction of Melanoma Cells and

Endothelial cells.

Previous irnrnunofluorescence staining using the rnAb GC4 raised against chick

N-cadherin failed to detect N-cadherin in the heterotypic contacts, although rnAb GC4

exerted partial inhibition on the transendothelial migration of melanoma cells (Sandig et

ai., 1997). To funher investigate the role of N-cadhenn in melanoma transmigration,

antibodies from several commercial sources were examined in immunofluorescence

labeling studies. The anti-N-cadherin mAb purchased from Transduction Laboratory

showed the strongest N-cadhenn signai and was used in subsequent expenments.

Melanoma cells were pre-labeled with red ce11 tracker dye and then seeded on top of a

monolayer of HMVEC. The CO-cultures were fixed at 1,3 and 5 h and stained using the

N-cadhenn mAb for confocal rnicroscopy. Fig. 8A shows a series of optical sections

through a melanoma ce11 intercalated between endothelial cells. N-cadherin staining was

evident in the contact regions between the melanoma ce11 and its surrounding endotheliai

cells. N-cadherin was present from the apical region to the basal region of the heterotypic

contacts.

For better visuaiization of the transmigrating melanoma cells, 3-dimensional

reconstruction of confocal series was conducted (Fig. 8B). N-cadherin was diffusely

distributed on the surface of the endothelial cells and it was not concentrated in the

interendothelial junctions. In contrast. an ennchment of N-cadherin was observed in the

heterotypic contacts with the melanoma cells (Fig. 9). At initial stages when melanoma

ce11 attached to endothelial monolayer and was induced to send out membrane

protrusions in the basolateral regions, intense staining of N-cadherin was frequently

observed ai the tip of these promsions. As the melanoma ce11 began to invade the

endotheliai junction by inserting pseudopodia between endothelial cells, a higher

concentration of N-cadherin was present in the heterotypic contact regions. At the later

stages of transmigration when melanoma cells became intercdated between endothelial

cells, predorninant N-cadherin staining was associated with the heterotypic contacts and

the leading edge of surrounding endothelial cells spreading on top of the melanoma cells

(Fig. 10). These data thus suggest the direct involvement of N-cadherin throughout

melanoma transendothelial migration.

Lack of N-cadherin Mediated Contacts between WM35 Cells and Endotheliai Cells

The above results suggested that N-cadherin was redistributed to heterotypic

contacts after the dissolution of the inter-endothelid adhesion complexes and that it

might play a critical rote during transendothelid migration of melanoma cells. It was.

therefore, of interest to examine the distribution of N-cadherin in the non-metastatic ce11

line WM35. WM35 was established from the radial growth phase of melanoma

progression and WM35 cells undergo transendothelial migration poorly in the CO-culture

assay (Voura et al.. l998a). When seeded on top of HMVEC, WM35 cells attached well

but only 10% of them were found to complete transmigration in 5 h. When the CO-

cultures were stained with mAb against N-cadhenn, most of the WM35 cells showed

negative staining at heterotypic contacts (Fig. 1 1 A). in contrast, N-cadherin-mediated

contacts could be observed in the homotypic contact regions between WM35 cells (Fig.

1 1B). Only -20% of WM35 cells showed N-cadherin staining when CO-cultured with

endothelial cells compared to 85% of WM239 cells (Fig. 1 1C).

WM35 Cells Express an Abnonnally Glycosylated Form of N-cadherin

Western blot analysis was carried out to determine whether the inability of WM35

cells to form heterotypic N-cadherin contacts was due to a lower level of N-cadherin

expression. The results showed that the level of N-cadherin expressed in WM35 cells was

similar to that in WM239 cells (Fig. 12A). However, the N-cadherin expressed in WM35

cells reproducibly migrated slightly faster than the N-cadherin derived from either

WM239 cells or HMVEC. The difference in molecuiar size was estimated to be 4-5 ma.

This difference in molecular weight might be due to differences in posttranslational

modification. such as glycosylation. To test this hypothesis, both WM239 and WM35

cells were treated with the N-glycosylation inhibitor tunicamycin. After incubation in 5

pg/mi of tunicarnycin for 12 h, a lower molecular weight form of N-cadherin was

detected in WM239 cells and it migrated tu the same position as the N-cadherin species

found in WM35 cells (Fig. 12B). This result suggested that the difference is due to an

alteration of N-glycosylation in WM35 cells.

Inhibition of the Expression of N-cadherin Using Antisense Approaches

To determine whether N-cadhenn plays an important role in the extravasation

process of melanoma cells, 1 tested whether the reduction of N-cadherin expression might

inhibit melanoma ce11 transendothelial migration. My initiai attempt was to use antisense

oligonucleotides purchased from Biognostik to inhibit N-cadherin expression. WM239

cells were incubated with the recommended dose of 2 rnM antisense oligonucleotides for

1,2,4,8,24 and 48 h. However, there was no detectable inhibition in N-cadherin

expression at 48 h. Funher attempts included doubling the oligonucleotide concentration

and the incubation tirne but again no inhibitory effect was observed even at 96 h (Fig.

13A). To rnonitor the cellular uptake of oligonucleotides, WM239 cells were incubated

with fluorescence-labeled ~Iigonucleotide (Fig. 13B). Aithough antisense

oligonucleotides actually entered these cells, the level of Ntadherin was not reduced

significantly.

After the unsuccessful attempt of using commercial oligonucleotides, a second

approach was conducted to express antisense oligonucleotides in cells by DNA

transfection. A plasmid carrying an N-cadherin antisense sequence was introduced into

WM239 cells (Fig. 5). A modified human U6 snRNA gene containing a 25-bp antisense

fragment flanking the start codon of human N-cadherin was cloned was used to express

the antisense sequence (Islam et al.. 1996). The U6 snRNA cm be synthesized

constitutively at high levels, up to 5 x 106 copies per ce11 regardless of celi types

(Noonberg et al., 1994). WM239 cells were transfected using the calcium phosphate

precipitation method (Chen and Okayama, 1987). Transfectants were selected using

puromycin at the initial concentration of 0.5 p g h l for 4 weeks. The puromycin-resistant

colonies were isolated and expanded. Colonies were isolated using cloning rings and then

cultured in 35-mm dishes to confluence. A total of 44 stable clones were expanded and

tested for N-cadhenn expression by Western blot analysis (Fig. 14). Among them. the

clones A 12 and A35 showed the greatest reduction in N-cadhenn expression.

Next, in vitro transendothelial migration assays were carried out using the A 12 and

A35 cells. The percentage of transfected cells that could undergo transendothelial

migration was scored at 1 ,3 and 5 h of co-culture. WM239 cells and clone A2, which

expressed the sarne level of Ntadherin as the parental cells. were used as controls. Both

A12 and A35 cells showed -30% reduction in transendothelial migration, whereas the

rates of transmigration were similar for both A2 and WM239 celis (Fig. 15). These

results suggest that Ncadhenn is important to melanoma ce11 transendothelial migration.

However, additional factors may also be involved.

Absence of p-catenin at the Heterotypic Contact Regions between Melanoma Cells

and Endothelial Cells

The cytoplasrnic tail of classical cadherins is known to associate with catenins. The

localization of N-cadherin in the heterotypic contacts should predict the CO-localization of

catenins in the same region. When coîultures were labeled with rnAb against p-catenin,

I was surprised to find that a higher concentration of B-catenin was not detected in the

heterotypic contacts. Dissolution of p-catenin underneaih the melanoma ce11 was

observed upon adhesion of melanoma ceil to the endothelium (Fig. 16A, a. arrowhead).

p-catenin was either absent or present diffusely in the heterotypic contact regions

between transmigrating melanoma cells and endothelial cells (Fig. 16A, b, d, and d.

arrowheads). p-catenin staining was only present in the melanoma blebs and leading edge

of the endothelial protrtssions spreading on top of melanoma cells (Fig. 16A, a', b'. c', and

d', arrows). In contrast, a clear staining pattern similar to that of VE-cadherin was

observed at the interendothelial junctions for p-catenin (Fig. 16 A, a, arrow). As a

control, melanoma cells were stained for p-catenin and a higher concentration of P-

catenin was present in the cell-ce11 contact region (Fig. 16B).

Changes in the Profile of Roteins Associated with N-cadherin

Cytoplasrnic proteins in the cadherîn complex are known to play important roles in

regulating the binding specificity, and the adhesive strength of cadherins (Yap et al.,

1997). It was, therefore, of interest to identiS components associated with Ntadhenn in

the heterotypic contacts between melanoma cells and endothelial cells. To identify

changes of the cytoplasrnic proteins associated with N-cadherin, imrnunoprecipitation

was carried out using rabbit anti-N-cadherin antibody to pull down proteins associated

with Ntadherin in ce11 lysates before and after CO-culturing. Silver staining revealed

changes in the gel profile between precipitates collected from CO-cultures and rhose from

a mixture of the two ce11 types. Several new bands were present in the CO-culture sample

(Fig. 17A). The most prominent change was the appearance of a band at 42 kDa, a

molecular weight corresponding to that of actin. Its identity was confirmed by

irnmunoblotting with an anti-actin rnAb (Fig. 17B). The data suggested that interactions

between melanorna cells and endothelial cells led to the association of the cytoskeleton

with the N-cadherin cornplex.

Part II: Role of Gap Junctions during Transendothelial Migration of

Melanoma cells

Gap Junctions in Heterotypic Contacts between Melanoma Cells and Endothelial

Cells

in addition to N-cadherin, the role of gap junctions during diapedesis of melanoma

cells was exarnined. Coîultures of WM239 cells and HMVEC were stained using

antibodies against the gap junction protein connexin43. A punctate staining pattern of

connexin43 was observed dong the contact surfaces between transmigrating melanorna

cells and endothelial cells. However, connexin43 staining was much less frequent

between the non-metastatic WM35 cells and endothelial cells (Fig. 18A). To quantify the

formation of gap junctions during transendothelial migration of melanoma cells, the

connexin43- positive spots in the heterotypic contacts were counted. When scoring for

WM35 cells, they were separated into two groups: (1) cells undergoing transmigration,

and (2) cells spreading on top of endothelium. The metastatic WM239 melanoma cells

formed > 2-fold more gap junctions with endothelial cells than the non-transmigrating

W M 3 5 cells (Fig. 18B). However, among the small number of WM35 cells that were

able to undergo transmigration. they formed gap junctions with endothelial cells at a level

similar to that of WM239 ceIls.

Effects of 1-Heptonal on Melanorna Transendothelial Migration

The role of gap junctions was further examined using the gap junction inhibitor 1-

heptanol (Kimura et al., 1995). The addition of 3.5 mM heptanol in the medium resulted

in -50% reduction of melanoma ce11 transmigration (Fig. 19). These data thus suggest

that cell-ce11 communication via soluble factors between mehoma celIs and the

endothelium may play an important role during transmigration of tumor cells.

Fig. 6. Cadherins expressed in melanoma cells and endothelial cells. (A) Equal arnount (20

pg) of whole ce11 lysates of WM239, WM35 and HMVEC were separated by SDS-PAGE and

the protein blots were probed using N-cadherin, E-cadherin, P-cadhenn and VE-cadhenn mAb.

respectively. (B) Confocal images showing the immünofluorescence labeling pattern of N-

cadherin, E-cadherin and P-cadherin in WM239 cells, which were stained individually with

rnAb against Ncadherin, E-cadherin or P-cadherin. Bars = 10 p.

Fig. 7. Increase in the level of N-cadherin in CO-cultures of melanoma cells and

endothelial cek. (A) Immunoblot profiles of N-cadherin and actin of various ce11

samples. Whole ce11 lysates from single cultures and cocultures were tested for N-

cadherin expression. (B) Increase in N-cadherin level in CO-cultures. The relative amount

of N-cadhenn was quantified and the pixel values were normalized to that of actin. Lanes

1 and 2. Iane 3 and 4 contained the same number ( 1.2 x 106) of WM239 cells and

HMVEC, respectively. Lane 5 is a mixture of equal arnounts of cell lysate from 2 and 4:

lanes 6 and 7 are co-cultures of WM239 cells on HMVEC taken at 3 h and 5 h,

respective1 y.

1. WM239 on plastic dish 2. WM239 on Matrigel 3. Endothelial cell on plastic dish 4 Endothclial cell on Matrigel 5. Mixture of equal amount of lysatc from 2 and 3 6. 3 hours ca-culture 7. 5 hours co-culture

Lane 5 Lane 6 Lane 7

.. . 6 1.5 - v .-*.. -r ........

6 . - . . . ........ z , ........ ....... ........ ....... '=. ........ ....... m . . . . . . . ....... 2 1 - . . J i ........ .. . . . . . . . . . . . . . . 3 B . . . . . . . n . . . . . . . . . . . . . . . . . . . . . . . > ........ ........ ................ ........ 3 . . . . . . .a . . .............. - ........ ....... ..-.... ........ L3 0.5 - ;:::::::::::I::: : ....*... ....... > ........ b . . . . . . . ........ .......

b . . . . . . . ........ d ....... ........ ................ ........ ....... . . . . . . . . . . . . . . . . ........ ....... ....... ........

........ ........ ........ ........ ........ ........ ........ b.. . . . . . ........ . ........ b . . . . . . ........ ........ b....... ........ ........ ........ ........ ........ ........ ........ ........ ........ b....... ........ ........ ........ ........

.. . . . .a . ........

. . a * . . . . ........ .-...... ........ ........ I

Fig. 8. Confocal images showing N-cadherin localization in the heterotypic

contacts between endothelial cells and a transmigrating melanoma cell. (A)

WM239 melanoma cells were labeled with red ce11 tracker before seeding on a

HMVEC monolayer. Co-cultures were fixed using methanol and stained with N-

cadhenn mAb. A series of optical sections (0.4 p thick) in the apical ( a ) to basal fl

direction shows the transmgration of a melanoma ce11 through a HMVEC monolayer.

N-cadherin (green) is enriched in the heterotypic contacts between melanoma ceIl and

its surrounding endothelial cells (arrowheads) and at the tip of a pseudopod

penetrating endothelial rnonolayer (arrows). (B) 3-D reconstruction of the series of

optical sections shown in (A). The melanoma ce11 is shown in red. Green represents

N-cadherin staining (g). The merged picture of g is shown in g'. Bar = 10 Pm.

N-cadherin Merge

56

Fig. 9. Formation of N-cadherin-rich heterotypic contacts between melanoma ce11 and

endothelial cells in the early stages of melanoma transendothelial migration. These

images represent the 3-D reconstruction from two series of opticai sections of melanoma

cells in their early stages of transmigration. Melanoma cells were pre-stained with red cell-

tracker (red) before seeding on the HMVEC monolayer. The CO-cultures were labeled with

N-cadhenn mAb (a, b, green). The merged images of a and b are shown in n ' and h' .

respectively. In (a) , the 2-projection viewed from the apical side shows that the melanoma

ce11 was on top of the HMVEC monolayer. This melanoma ce11 began to extend membrane

protmsions on the endothelium and N-cadherin staining was observed at the tip of thrse

protmsions (arrowheads). In (b). the 2-projection is viewed from the ventral side. which

shows the pseudopod of a melanoma ce11 intercdated in the endothelial junction and the

enric hmen t of N-cadherin at the heterotypic contact regions (nrroivhends). Schematic

drawings depicting the stage of transmigration are show on the left Bars = 10 Fm.

N-cadherin Merge

Fig. 10. Formation of N-cadherin-rich heterotypic contacts between melanorna cells and

endothelial cells in the late stages of transmigration. These images represent the 3-D

reconstruction from two series of opticai sections viewed from the apical side. depicting the

late stages of transendothelial migration by melanoma cells. Melanoma cells were pre-stained

with red cell-tracker. The CO-culture was labeled with rnAb against N-cadherin ( t ~ . b, green).

The merged images of a and b are shown in a ' and b'. respectively. in ( a ) a melanoma ce11

intercaiated between endothelial cells. in (b), the endothelial cells began to spread over the

melanoma cell, which was also spreading on the Matrigel. N-cadherin enrichment was

observed at the heterotypic contact regions (a. b, arrows) and the tips of pseudopod ((1. h.

arrowheads). Bars = 10 Pm.

N-cad herin Merge

Fig. 11. Lack of N-cadherin enrichment in the heterotypic contacts between WM35

melanoma cells and endothelial cells. (A) Confocal images of CO-cultures of (a) WM35 ce11

and (b) WM239 ce11 seeded on a HMVEC monolayer. Staining of N-cadherin at the

heterotypic contacts of WM239 ce11 with HMVEC was significant (arrowheads). whereas

such staining was rarely observed between WM35 ce11 and HMVEC. Melanoma cells are

indicated by "m". (B) immunostaining of N-cadherin in the cell-ce11 contact region between

WM35 cells (arrowheads). Bars = 10 Pm. (C) The percentage of melanoma cells that formed

N-cadherin-Rch contact with HMVEC was estimated. A total of 40 cells were scored in each

assay.

WM239 W M 3 5 Wh4239 W M 3 5

- tunicamycin + tunicamycin

Fig. 12. Expression of an N-cadherin variant in WM35 melanoma cells. (A)

Total ce11 proteins of WM239 cells and WM35 cells were separated by SDS-PAGE

and the protein blot was pmbed with mAb against N-cadherin. (B) Immunoblot of N-

cadherin after WM239 and WM35 cells were treated for 12 h with 5 pgiml of

tunicarny c in.

Control oligonucleotides (4 mM) Antisensc oligonucleotides (4 mM)

I w

f- Actin

-

4 8 24 48 72 96 4 8 23 48 73 96 Time (h)

Fig. 13. The antkense oligonucleotides fmm Biognostik did not inhibit N-cadherin

Expression in Wh4239 cells. (A) WM239 cells were treated with either antisense or control

o1igo:onucleotides at a concentration of 4 m M for 4,8,24,48,72 and 96 h. Western blot

analysis was performed to masure the expression of N-cadherin and actin at each time

period. (B) Uptake of antisense oligonucleotides by WM239 cells was monitored using FITC-

labeled oligonucleotides. Epifluorescence micrographs of WM239 cells after 4 h and 96 h of

incubation with 4 mM oligonucleotides are shown in a and b. Bars = 20 ym.

Fig. 14. Effects of the transfection of an N-cadherin antisense construct on the

expression of N-cadherin In WM239 cells. WM239 cells were transfected with the

pBSpacAp' plasmid and purornycin-resistant colonies were isolated and expanded. (A)

Western blot analysis was cmied out to examine the expression of N-cadherin in these

transfectants. Total ceil proteins were subjected to SDS-PAGE and the protein blots were

probed with mAb against N-cadherin and actin. (B) The relative amounts of N-cadherin

expressed in these transfectants. The imrnunoblots were quantified and the pixel values

were fiat normaiized to that of actin and then normalized to that of the parental WM239

cells. Clones A 12 and A35 (indicated by an asterisk) showed the iowest level of N-

cadherin.

Time (h)

Fig. 15. Effects of reductioo in N-cadherin expression on the transendothelial

migration of melanoma cells. Clones transfected with the N-cadherin antisense consuuct

were examined in the M vitro tramendothelid migration essay. Clones A12 and A35 had a

drastic reduction in N-cadherin expression while the level of N-cadherin in clone A2 (i)

was sirnilar to that of WM239 cells (+). Co-cultures were carried out for 1,3. and 5 h

before fixation and ceils were stained with mAb to visualize F-actin in order to facilitate

the scoring of transmigrated cells.

Fig. 16. Lack of p-catenin enrichment in the heterotypic contact regions between

melanoma cells and endothelial cells. Melanoma cells were pre-stained with red cell-tracker

before seeding on a HMVEC monolayer. The CO-culture was labeled with mAb against B-

catenin (green). Panels a*, b'. c'. and d' represent optical sections 1.2 p. 2.4 prn, 1.8 pm.

and 2.4 p above (apical side) those shown in panels a, b, c. and d respectively. In (a), a

melanoma ce11 was in the initial stage of transendothelial migration. p-catenin started to

redistribute away from the region undemeath the melanoma ce11 (a. arrowhead), whereas

endothelial junctions outside the contact region remained intact (a, arrow). In (a'), weak P-

catenin staining was associated with membrane protrusion in the heterotypic contact (a',

mow). In (b). a melanoma ce11 was intercalated between two endotheliai cells. p-catenin was

absent in the heterotypic contact regions (b, arrowheads). However, p-catenin staining was

evident in the leading edge of an endothelial cell. which began to spread on top of melanoma

ce11 (b'. arrows). In (c). the melanoma ce11 was spreading on the Matrigel. Sirnilar to (b), B-

catenin was absent in heterotypic contacts (c. arrowheads), but was concentrated in the

leading edge of endothelial cells re-closing the gap caused by the melanoma ce11 (c', arrows).

In (d), the melanoma ce11 has completed transmigration and adopted a fibroblastic

morphology on the Matrigel. No p-catenin was observed in the heterotypic contact regions

between the melanorna cell and endothelial cells (d, arrowhead). However. strong p-catenin

staining was found in the inter-endothelid contacts reestablished on top of the melanoma ce11

(d', arrow). (B) As a control, WM239 cells were stained with Btatenin rnAb and prominent

staining of ptatenin was present along the melanoma cell-ceil contact regions (arrow). Bars =

10 W.

Fig. 17. Changes in the profile of proteins associated with N-cadherin. Soluble proteins were

prepared from the single ce11 cultures and CO-cultures by extraction of ce11 with lysis solution.

Immunoprecipitation was can-ied out using rabbit anti-N-cadherin antibody to pull down proteins

associated with N-cadherin. (A) Silver staining patterns of the different immunoprecipitates.

New protein bands in precipitates collected from the 5-h CO-culture are marked with arrows on

the right. Molecular weight markers are shown on the left. (B) Identification of the 42 kDa band

as actin. Proteins in the gel were transferred to a nitrocellulose membrane and then probed with

anti-actin mAb.

WM239 cells HMVEC Mixture of lane 1 and lane 2 5 h CO-culture

Fig. 18. Imrnunolocalization of gap junctions in the heterotypic contact regions during

transendothelial migration of melanoma cells. WM239 melanorna cells were pre-labeled

with Di1 (red) and then seeded on top of a HMVEC monolayer. The CO-cultures were fixed

at 5 h and then stained with anti-connexi1143 mAb (green). (A) The interendothelid contact

regions were decorated with punctate connexin43 staining (arrorvs). Gap junction staining

was also evident dong the heterotypic contact reg ions between the transmigrating melanoma

ceIl and its surrounding endothelid cells (nrrowheods). (B) Quantification of the number of

connexin-stained gap junction complexes in the heterotypic contact regions of WM239 and

WM35 cells. The WM35 cells were separated into two groups according to whether or not

they were penetrating the endothelium based on confocal microscopy. Thiny cells wcre

analyzed for each catrgory. The number of "dots" was counted and the length of the

heterotypic contacts was rneasured usinp a cuwimeter. Bar = 10 Pm.

Number of connexin43-positive dots per 100 Pm

I h 3 h 5 h

control

a 1 -heptano1 treatment

Fig. 19. Inhibition of transendothelial migration of melanoma cells is

inhibited by the gap junction inhibitor 1-heptanol. 1 -heptano1 was added at

a concentration of 3.5 mM. The CO-cultures were fixed at 1.3 and 5 h and then

stained for F-actin. The number of transmigrated cells was scored for each time

point.

Chapter 4: Discussion

Involvement of N-cadherin in Melanoma Transendothelial Migration

In my thesis project, 1 have carried out experiments to examine the morphologicai

and biochemical aspects of the involvement of Ncadherin during melanoma

transendothelial migration. To test the hypothesis that the classical cadherin that becornes

concentrated in the heterotypic contacts during transmigration of melanoma cells is N-

cadherin. several mAbs against N-cadherin were screened for their ability to recognize N-

cadherin in both immunoblots and fixed cells. The anti-N-cadherin mAb purchased from

Transduction Laboratory proved to be most useful. Imrnunolocalization studies

demonstrated that N-cadherin was concentrated in the heterotypic contacts between a

WM239 cells and endothelial cells at al1 stages of transmigration. These results correlated

with our earlier inhibition studies using mAb GC4 (Sandig et al., 1997), which was raised

against chick A-CAM, a homologue of human and mouse N-cadherin (Volk and Geiger,

During embryonic development, neural crest cells, the precursor of melanocytes,

express N-cadherin predominantly (Weston, 1970; Ayer-Le Lievre and Fontaine-Perus,

1982). Neural crest cells migrate away from the neural tube and differentiate into many -

different ce11 types, including melanocytes. Expression of N-cadherin in melanocytes is

replaced by E-cadherin when they become dormant in terms of motility (Nakagawa and

Takeichi, 1998). Re-establishment of the expression of N-cadhenn in melanocytes may

be the result of dedifferentiation and enables the invasion of neighboring tissues by the

transformed melanoma cells. While N-cadherin is upregulated, E-cadherin is down-

regulated in melanoma cells (Hsu et al., 1996). Reduction or loss of Etadherin

expression has been well-documented in various cancers, especially in those derived from

the epithelium (Behrens, 1994). E-cadherin is the major adhesion molecule present

between melanocytes and keratinocytes, which may regulate the proliferation of

melanocytes (Tang et al., 1994). It is hypothesized that by reducing E-cadherin

expression while increasing N-cadherin level, melanoma cells may escape the growth

regulation of surrounding keratinocytes, which express high levels of E-cadherin. When

melanoma cells invade into the deeper demis layer, they interact with fibroblasts that

express N-cadhenn (Fig. 20). Thus, the replacement of E-cadhenn with N-cadherin may

facilitate the invasion of melanomas in their early stages of turnor development and

permit their unregulated proliferation.

The switching of E-cadhenn to N-cadherin does not occur only in melanoma.

There is increasing evidence that cadhenn switching plays a key role in cancer

progression, ce11 motility and metastatic potential of malignant cells (Islam et al., 1996;

Hazan et al., 2000). Similar phenornena have been observed in human prostate carcinoma

(Tornita et al., 2000), anaplastic thyroid-carcinoma (Husmark et al., 1999), human

squamous carcinoma (Islam et al., 1996), and malignant T-ce11 lyrnphoma (Kawarnura-

Kodama et al., 1999). The mechanisms that regulate cadherin expression are not known.

It is hypothesized that E-cadherin and N-cadherin may be inversely regulated in cells

(Islam et al., 1996).

The data presented in this thesis point to a novel role of N-cadherin in the

extravasation step during cancer metastasis. Mature endothelium expresses VE-cadhenn

and N-cadherin (Dejana, 1997). However, oniy VEcadherin is localized in the

endothelial cell-ce11 contact region (Navarro et al., 1998). Ncadherin is excluded from

the endothelial junction by VEcadherin (Navarro et ai., 1998). Melanoma cells detached

Normal melanoc yte

LI E-cadherin-mediated adhesion L N-cadherin mediated adhesion

Fig. 20. Switching of E-cadherin to N-cadherin accompanying melanoma

progression. Normal epidermal melanoc ytes interact with adjacent keratinoc ytes

through E-cadhenn-rnediated adhesion. After the expression of E-cadherin is switched

to N-cadherin, melanoma cells migrate into the deeper dermis Iayer and interact with

fibroblasts (Hsu et al., 1996).

from the primary foci travel in the circulatory system and may get trapped at

microvascular blood vessels due to mechanical force or interaction with specific ce11

surface moIecules of the endotheliurn in the target organ. After melanoma cells attach to

the apical surface of the endothelium, N-cadherin from both ce11 types becomes

concentrated in the heterotypic contacts. At the site of melanoma ce11 transmigration,

cell-ce11 adhesion complexes between endothelial cells start to dissociate, allowing the

passage of melanoma cells (Voura et ai., 1998b). Notably, PECAM- 1 in the endothelial

junctions beneath a melanoma ce11 redistributes to other parts of the ce11 (Voura et al.,

1998a). Sirnilarly, adherens junctions begin to dissociate and VE-cadherin redistributes to

other regions of the endothelial ce11 membrane (Sandig et al.. 1997). N-cadhenn may now

move in and occupy the area left behind by VE-cadherin. Dunng the transmigration

process, the N-cadhenn-based contact may facilitate the migration of melanoma cells on

the endothelial ce11 surface. Recent studies have suggested that N-cadherin might

promote ce11 motility and invasion in cancer cells (Nieman et al., 1999; Hazan et al.,

2000). Thus, it is possible that N-cadherin hnctions in the transendotheliaî migration of

cancer cells.

Although the cytoplasmic domain of classical cadherins is known to associate with

actin filaments via p-catenin and a-catenin (Yap et al.. 1997), it is surprising that P-

catenin is either present in very low levels or absent in the heterotypic contacts. Since

strong cadhenn-mediated ce11 adhesion is dependent on its association with p-catenin

(Aberle et al., 1996), the lack of p-catenin in the heterotypic contacts suggests weak N-

cadherin-mediated adhesion between the transrnigrating melanoma ce11 and its

surrounding endothelial cells. The weaker adhesive force may facilitate ce11 migration.

Upon the completion of transmigration, melanoma cells spread undemeath the

endothelium and begin to invade the extracellular matrix.

Potentiat Role of N-glycosylation on N-cadherin Function

The ce11 line WM35, which was established from the radial growth phase of

melanoma (Herlyn et al.. 1985). undergoes transmigration poorly in the in vitro CO-

culture assay (Voura et al., 1998a). Despite the expression of a sirnilar level of N-

cadherin, only a small percentage of WM3S cells can form N-cadherin-mediated cell-ce11

contacts with endothelid cells. Interestingly, WM35 cells express an N-cadherin species

with a slightly lower molecular weight, which is estimated to be 125 kDa. In comparison.

the mature N-cadhenn molecule of WM239 cells and HMVEC migrated at 130 kDa.

Using the N-glycosylation inhibitor tunicamycin, 1 have found that a new species of N-

cadherin of 125 kDa begins to accumulate in WM239 cells, suggesting that the lower

molecular weight fonn expressed in WM35 cells may be the result of aberrant N-

glycosylation of N-cadherin. Abnormal glycosylation is one of the major molecular

changes known to accompany mdignant transformation (Dennis et al., 1999). Severd

studies have been carried out on the effects of N-giycosylation on the function of

cadherins. For instance, increased glycosylation of E-cadhenn in B 16 murine melanoma

cells can lead to the suppression of tumor metastasis (Yoshimura et al., 1996). Although

the elevated glycosylation does not influence the expression of E-cadherïn, it does have

an effect on the turnover of E-cadhenn, resuiting in increasing membrane distribution.

My results suggest that the glycosylation state of N-cadherin rnay have an influence on

the metastatic potential of melanoma cells.

WM35 cells may express an N-cadherin species with altered N-glycosylation,

such that they can no longer form N-cadherin-based contacts with endothelial cells. It is

of interest to test whether this is the reason for the poor transmigration of WM35 cells in

the CO-culture assay. It is possible that alteration of N-glycosylation may have an effect

on the conformation of N-cadhenn and indirectly alter the homophilic binding affinity of

N-cadherin in WM35 cells. This in turn may have an adverse effect on the

transendothelial migration process.

Effect of N-cadherin Antisense Oligonucleotides on Transendothelial Migration of

Melanoma cells

To further investigate the role of N-cadherin in melanoma transendothelial

migration, the antisense approach was employed to block the expression of N-cadherin.

Initially, commercially available oligonucleotides were tested. However, no significant

reduction in N-cadherin expression level could be detected even with higher

concentrations and extended incubation periods. Observations with the fîuorescence-

labeled control oligonucleotides indicated that the Wh4239 cells did take up these

oligonucleotides. The lack of inhibitory effects might be due to the low efficiency of the

oligonucleotides to reach the cytosol. The control oligonucleotides showed punctate

staining within the cells, suggesting that they might still be sequestered in vesicles in the

cytoplasm. Thus, the effective concentration of oligonucleotides within the ce11 might not

be enough to interfere with the expression of N-cadherin.

Inhibition of N-cadherin expression of in WM239 cells was achieved by another

approach. Cells were transfected with the N-cadherin antisense construct. Two

transfectants, A12 and A35 showed the lowest level of N-cadherin expression. However,

only 30% inhibition of transendothelial migration was observed in the CO-culture assay.

One possible explanation is that even though N-cadherin is greatly reduced, the

remaining amount is sufficient to facilitate the transmigration of melanoma cells. To

adàress this problem, selection of transfectants with no N-cadherin expression is

currently being attempted by stepwise increase in purornycin in the selection medium,

which may lead to amplification of the integrated plasrnid DNA and a higher level of

antisense RNA.

Not al1 cancer cells express Ntadhenn. in the case of melanoma cells, only

partial inhibition has been achieved by either antibody or antisense approach. It is

therefore likely that other ce11 adhesion molecules also play important roles in the

transendothelial migration of melanoma cells. Recent results obtained in Our laboratory

have demonstrated that the heterophilic binding between L1 and integrin is involved

in the transendothelial migration of melanoma cells (Voura, 1999). Another candidate is

the MUC18 protein, which is a maker of melanoma cells (Shih et al., 1994). MUC 18 is

an Ig-CAM that mediates cell-ce11 adhesion via binding with a heterophilic receptor (Shih

et al., 1997). The level of MUC 18 has been shown to correlate with the metastatic

potential of melanoma cells. In addition, melanoma cells express the ligand of MUC 18

(Shih et al., 1997). Endothelid cells also express an abundance of MUC 18 molecules on

the ce11 surface. It is, therefore, possible that transmigration of melanoma cells may

involve MUC 18.

Changes in the Profile of Proteins Associatecl with N-cadherin

The immunoprecipitation results clearly show that, upon interaction between

melanoma cells and endothelial cells, actin becomes associated with N-cadherin. This

observation correlates well with previous data in our laboratory that acun filaments are

sent out perpendicular to the heterotypic contact regions between tumor cells and

endothelial cells (Voura et al.. 1998b). In endothelial cells, N-cadherin is distributed

diffusely on the ce11 membrane and is not involved in intercellular interaction. Thus, the

association of N-cadherin and cytoskeleton is marginal. During melanoma

transmigration, however, N-cadhenn is recruited to the heterotypic contacts and may

Function in promoting the migration of melanoma cells. Therefore, the association of N-

cadhenn with the cytoskeleton becomes necessary.

How is N-cadherin linked to the cytoskeleton? p-catenin is an important

component of the well-established cadherin complexes and is known to associate with

VE-cadherin complexes between endothelial cells (Dejana et al., 1999). In contrat, only

diffuse B-catenin staining is present in the Ncadherin-enriched contacts between

melanoma cells and endothelial cells. This observation suggests that the VE-cadherin

complexes in the endothelial junctions and the N-cadhenn complexes in the heterotypic

contacts are different in nature. The VE-cadherin complexes with B-catenin association

may represent stable adherens junctions that maintain strong cell-ce11 adhesion (Bullions

and Levine, 1998). whereas the heterotypic N-cadherin contacts may favor less stable and

weak cell-ce11 adhesion between tumor cells and endothelial cells. Compared to the VE-

cadherin complexes, N-cadherin complexes in the heterotypic contacts may be transient

dynamic stmctures. It will be of interest to determine which molecule serves as a linker

between N-cadherin and the cytoskeleton in this case.

One possible candidate is p l2OCtn, which is, currently, rnainly known as a

regulator of cadherin complexes by its phosphorylation state (Yap et al., 1998). p 1 2OCm

binds to the membrane-proximal region of the cytoplasmic domain of classical cadherins

(Yap et al.. 1998). Even though evidence of direct association of p120C" with

cytoskeleton is still lacking, it is possible that p12OCm interacts with cytoskeleton via a

mechanism that does not involve a-catenin. There are several proteins in the cadherin

complex that are known to bind actin, such as 20-1 (Itoh et al., 1993) and vinculin

(Hazan et al., 1997). Potentially. these molecules may provide an alternative mechanism

that leads to the connection between N-cadhenn and the cytoskeleton at the heterotypic

contacts between melanoma cells and endotheliai cells.

Another role of N-cadherin in melanoma transendothelial migration is the

mediation of endotheliai ce11 spreading on top of the transmigrated melanoma cells. As

shown in the Results section, b-catenin was present in the leading edge of the re-closing

endothelial cells on top of melanoma cells, suggesting a different role of N-cadherin other

then the weak heterotypic interaction, which facilitates the passage of tumor cell. The N-

cadherin complex contains p-catenin in those apical regions, confemng stronger adhesive

interaction, and may provide the necessary traction force for the spreading of endothelial

protmsions on top of melanoma cells.

Role of Gap Junctions in Melanorna Transendothelial Migration

Previous results from Our laboratory and other research groups suggest that the

endothelium serves more than a barrier during tumor ce11 extravasation (On et al.. 2000)

In fact, endothelial cells participate actively in cell-ce11 interactions during the

transmigration process. Cancer cells can sense the presence of endothelial cells and vice

versa. Signals are being transmitted ro regulate the morphology and behavior of both ce1

types. It is possible that cancer ce11 have adopted some of the mechanisms utilized by

leukocytes to "fool" the endothelial cells.

The signals exchanged between tumor cells and endothelial cells possibly include

contact-dependent signals and small signaling molecules. It has been shown that there is a

rapid and transient increase of intncellular ~ a " level in endotheliai cells upon contact

with tumor cells and it is thought to be a tyrosine kinase receptor-mediated phenomena

(Lewalle et al., 1998). Activation of tyrosine kinase may depend on the binding of

membrane molecules on tumor cells. The work included in my thesis has examined the

possible involvernent of gap junctions. which cm rnediate the exchange of small

signaling molecules dunng the melanoma transendothelial migration.

Gap junctions in the heterotypic contacts between transmigrating melanoma cells

and endothelial cells are just as abundant as those in the homotypic endothelial contact

regions. Also, incubation of c o t u l ~ r e s with 1-heptanol leads to a 50% reduction in

melanoma cells transmigration, suggesting that gap junctional communication plays a

role in this process. 1-Heptanol is a classic inhibitor of gap junctional communication

(Christ et ai., 1999), although the mechanisrn of inhibition is unclear. It is hypothesized

that 1-heptanol may interfere with the micro-lipid environment for the proper functioning

of gap junctions (Bastiaanse et al., 1993). The inhibitory effect of 1-heptanol is transient

and reversible, implicating a functional role of 1-heptanol rather than a structural role.

Small molecules of 4 2 0 0 Da, including some tumor metabolites cm go through

gap junctions freely. One of these tumor metabolites suggested to transfer via gap

junction from tumor cells to endothelial cells is 12(S)-hydroxyeicosatetraenoic acid

( 12(S)-HETE), which is a lipoxygenase metabolite of arachidonic acid. Expression of

11(S)-HETE by tumor cells correlates with the degree of experimental metastasis (Chen

et al., 1994). In addition, tumor cell production of 12(S)-HETE is increased upon contact

with the endothelium (Honn et al., 1994). I2(S)-HETE has been shown to induce

extensive responses in endotheliai cells, ranging from reorganization of cytoskeleton to

retraction of endotheliai ce11 junctions (Tang et al., 1993; Tang and Honn, 1994a).

Therefore, inhibition of gap junctional communication may block the signai exchange

between tumor cells and endothelial cells, leading to less efficient transmigration.

Cornparison of the Extravasation Process between Cancer Cells and Lymphocytes

Metastasis is the most convincing evidence of cancer. However, many aspects of

the metastatic behavior can be found in normal cells. For exarnpie, the migration of

leukocytes from the vascular system to sites of pathogenic exposure, which is a key event

in the process of inflammation, is similar to the hernatogenous dissemination of cancer

cells.

The extravasation of lymphocytes is initiated with rolling movement on the

endothelial cell surface. This rolling is largely mediated by selectins expressed by

lymphocytes and endotheliai cells. In contrast to the rapidly flowing cells in the blood

Stream, the rolling cells are able to sense signds from the endothelium. which may

stimulate them to adhere more firmly to endothelial ce11 surface. The selectin-mediated

rolling is followed by the activation of leukocyte integrins that can bind to Ig-CAM. The

major integrins involved in this process are LFA-1 (a&), Mac-1 and VLA4

(&pi). LFA- 1 and Mac- 1 cm bind to ICAM-1 whereas VLA4 may bind to VCAM- 1.

The integrin-mediated interactions implicate strong adhesion of lymphocytes to

endothelium (Lawrence and Springer, 199 1).

Al1 three classes of ce11 adhesion molecules, selectins. integrins and Ig-CAMs are

al1 reported to play roles in the extravasations of tumor cells. For example, it has been

reported that severai cancer ce11 lines also undergo selectin-mediated rolling (Giavazzi et

al., 1993; Fogel et al.. 1999). Integnns such as a& and Ig-CAM are also involved in the

interactions between cancer cells and endothelial cell. Compared to lymphocytes. cancer

cells express more diverse ce11 adhesion molecules. Therefore. cancer cells may make use

of ce11 adhesion moIecules other than those utilized by lymphocytes to undergo

extravasation. Results presented in this thesis show that N-cadhenn plays a key role in

melanoma transendotheliai migration. This phenornenon is so far unique to cancer cells.

Cancer cells and lymphocytes also share some common feature in the recognition

of sites of extravasation. Chemokines play important roles in the homing of lymphocytes.

About 40 known chemokines are able to specificdly activate certain leukocytes and

attract them to migrate across the endothelial barrier. The stimulatory effects of

chemokines cause activation of Ieukocyte integrins, thus leading to firm adhesion of

lymphocytes to endothelium. Similar to leukocytes, chemokines may have similar effects

on cancer cells (Rossi and Zlotnik. 2000). Tumor ce11 metastasis is known to be

facilitated by inflammation (Lafrenie et al., 1993). The homing of cancer cells also

involves the physical uapping and interaction between cancers and the ceIl adhesion

molecules expressed by endothelial cells. Al1 these observations suggest that cancer cells

have adopted many of the strategies utilized by lymphocytes during transendotheliai

migration, even through they may differ in some specific details.

Future Perspectives on the Study of Adhesive Interactions during Melanoma

Transendothelial Migration

As reviewed in the Introduction, ce11 adhesion is very important to the

maintenance of proper tissue morphology and interactions between neighboring cells. in

the case of extravasation, tumor cells interact in many ways with endotheliai cells as

depicted in our mode1 (Fig. 2 1). There is evidence that ce11 adhesion is important for the

establishment of cell-ce11 contacts and gap junctions. The gap-junctionai communication

between munne B 16 melanoma cells and lung endotheliai cells may depend on adhesion

mediated by Lu-ECAM- L (el-Sabban and Pauli, 1994). It has also been reported that

cadhenn junctions rnay influence the specificity of the formation of gap junctions. The

over-expression of E-cadherin in rat epithelial cells and rat fibroblasts leads to a 10-fold

increase in heterotypic communication via gap junctions (Prowse et al., 1997). in another

report, it is suggested that E-cadherin adherens junctions fomed between normal

melanocytes and surrounding keratinocytes ensure the formation of gap junctions

between these two ce11 types (Hsu et ai., 2000). When expression of E-cadherin is

switched to N-cadherin, melanoma cells interact with fibroblasts, thus facilitating the

formation gap junctions between these two cell types.

Fig. 21. A working mode1 depicting the role of N-cadherin in melanoma

transendotheiial migration. (A) At an early stage. the difisely distributed N-

cadhenn is recruited to cell-ceIl contacts between the melanoma ce11 and the

endotheliai cell. It is possible that N-cadherin facilitates the penetration of the

melanoma pseudopodia into the endothelium (a). In the later stages, in the absence

of p-catenin association, N-cadhenn provides a weak adhesive surface for the

penetration of melanoma cells and serves as a dnving force for the passage of these

cells. N-cadherin is also involved in mediating the spreading of endothelial

processes over the melanoma cells (b). When transmigration is completed.

melanoma cells start to spread on the Matrigel and VE-cadherin-mediated

endothelial junctions re-form on top of the melanoma cells (c). (B) Gap junctions

are involved in the interaction between tumor cells and endothelial cells. Exchange

of small signal molecules, such as iZ(S)-HETE, has been reponed to occur

between the two ce11 types possibly through gap junctions. The formation of gap

junction rnay require appropriate ce11 adhesion. especially cadherin junctions.

Therefore, N-cadherin mediated ce11 adhesion rnay facilitate the formation of gap

junctions, whch in turn support intercellular communication between tumor cells

and endothelial cells. Tumor metabolites may activate a signaling cascade in

endothelial cell, including a transient increase in intracellular ca2+ concentration.

which in turn may cause rearrangement of the actin cytoskeleton and dissolution of

endothelial cellcell contacts. Other adhesion-mediated signaling mechanisms rnay

also exist, such as the activation of protein tyrosine kinases in endothelid cells

upon the binding of turnor cells.

melanoma ce11

N-cadheri n VE-cadherin - b-catenin unknown molecule

-/ Actin Filaments

- m - m -

0 b

1 1

I

\

f

\

1

\

1

\

t

\

' Q Gap junction 1

1 1

f _---- __------- --- *--- I 0--

1 --. ': 0 PECAM- IlCD3 1

* - 0 - - . -. B j / - --. \ 12(S)-HETE

0'

; ,=' . Tyrosine kinase associated receptor 0 /

. ' '+, Proteolnic enzymes gnal molecules

Dunng the extravasation process, N-cadherin is involved in the ce11 adhesion

process between melanoma cells and endothelial cells. It is possible that the cadherin

complex may function as a scaffold for intercellular structures and facilitate the formation

of gap junctions. Thus, signal molecules are able to exchange efficiently between these

two ce11 types. Effects of these molecules could be extensive, such as the modification of

cell-ce11 adhesion between endothelial cells, which allows the passage of cancer cells. if

this is tme, the poor formation of gap junctions between WM35 cells and endothelial

cells could be the result of inefficient formation of N-cadherin based contacts, which may

also account for the poor transmigration of WM35 cells.

To address this issue, future experiments should be focused on the relationship

between N-cadhenn-rnediated heterotypic contacts and gap junction formation between

WM239 cells and HMVEC. Immuno-colocalization studies on connex43 and N-cadherin

in the heterotypic contact regions may provide evidence for their interaction. Besides

confocal observation of gap junction formation. the function of gap junctions can be

measured using Calcein AM in dye-transfer assays. which are cornrnonly used for

monitoring exchanges of small fluorescence molecules through gap junctions, (Juul et al..

2000). The ability of gap junction formation should be compared between WM239 cells

and WM239 cells transfected with N-cadherin antisense construct, such as the A12 and

A35 clones. These cells can be pre-loaded with Calcein AM and then seeded on the top

of a HMVEC monolayer. M e r 2-3 h of CO-culturing, the arnount of dye-transfer can be

monitored by the level of fluorescence in endothelial cells surrounding the transmigrating

melanoma cells as an indication of the function of gap junctions. If the above hypothesis

is m e , higher dye-transfer rate should be observed in WM239 cells than that in A12 or

A35 cells. It could also be of interest to test whether the formation of gap junction h a a

positive effect on the establishment of N-cadherin-rnediate ce11 contacts between

melanoma cells and endothelial cells. Experiments can be done to test whether the

inhibition of the functions of gap junction by 1-heptanol has any impact on the formation

of the heterotypic N-cadherin-mediated contacts and the dissolution of VE-cadherin

contacts between endothelial cells.

On the other hand. ce11 adhesion complexes themselves c m also take part in signal

transduction. in endothelid cells, only VE-cadherin is in its functional state, Le. forming

stable adhesive complexes. whereas N-cadherin is excluded from cell-ce11 contacts,

distributing diffusely on the ce11 surface. When melanoma cells and endothelial cells

establish N-cadhenn mediated ce11 contacts. it is expected that clustering of N-cadherin

molecules will lead to the recruitment of many cytoplasrnic molecules including

cytoskeleton components and signal molecules to N-cadherin-mediated adhesion

complex (Gumbiner, 1996). The information of new contacts cm be translated into the

remangement and dissolution of inter-endothelid ce11 adhesion complexes. such as those

formed by VE-cadherin and PECAM-1 (Voura et ai.. 1998b). After VEtadhenn

disappears between endothelial cells, endothelium becomes "leaky" to cancer cells. It is

possible that signais initiated by ce11 adhesion regulate the equilibrium of the VE-

cadhenn complex and N-cadherin complex through the cytosolic pool of p-catenin or

p12oCm. How are these two cadherins related and possible "cross-taiks" will be an

interesting topic for future study. With the development of new techniques such as high-

resolution 2-D electrophoresis and protein mass spectromeuy, it is possible now to

identifj proteins of very low amount (Pandey and Mann, 2000). As shown in the Results

section, new proteins are found to CO-imrnunoprecipitate with N-cadherin after melanoma

cells attach to HMVEC. These proteins are potential candidates as the components or

regulators of cadherin complexes. Their identities can be revealed by matrix-assisted

laser desorptiodionization (MALDI) spectrometry (Loo et al., 1999). Knowing the

identity of these proteins could provide extremely useful information for the

understanding the dynamic changes in the cadherin complex during tumor extravasation.

Another important experiment is to test whether the transendotheiial migration of

melanoma cells cm be inhibited by blocking both Ncadherin and a&. plays

important roles in the progression of melanoma and is the most specific melanoma-

associated marker that distinguishes VGP from RGP melanomas (Albelda et al., 1990).

Previous resulis in our laboratory have demonstrated that anti-a& rnAb and cyclic RGD

peptides inhibit rnelanoma transmigration by -50% (Voura, 1999). Since the integrin

a& and N-cadherin form different ce11 adhesion complexes, inhibition of these two

molecules may exert greater effects on the transmigration process. Incubation of A 12 and

A35 cells with anti-a& mAb or cyclic RGD peptides should reduce transmigration to

the background level. if promising results are obtained in these in vitro CO-culture assays,

the A 12 or A35 cells can be further transfected with ct,, or p3 antisense sequences.

Transfectants lacking both N-cadhenn and can be injected in nude mice to detemiine

the effects on the formation of expenmental metastases.

Cancers have been studied extensively for many years. Many different therapies

have been developed. such as chernotherapy, gene therapy and anti-angiogenesis dmgs.

Still, prevention of metastasis remains the most chailenging objective of a successhl

cancer therapy. If the adhesion of cancer cells to endothelium is inhibited, the danger of

cancer will be greatly reduced since cancer cells can be destroyed eventually by the

immune system and the shearing force in blood Stream (On et al., 2000). Although still at

an early stage of development, the research included in this thesis potentially cm provide

new clues for the control of metastasis.

References

Aberle, H., Butz, S., Siappert, J., Weissig, H., Kemler, R., and Hoschuetzky, H. (1994). Assembly of the cadhenn-catenin complex in vitro with recombinant proteins, J Ce11 Sci 107, 3655-63.

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