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uvomorulin expression, such as MCF-7, T47D, and MDA-. MB-i. 75-7. (Fig. 1 ,. a-c) showed ... be detected in cell lines MDA-MB- .... MDA-MB-468. -. +. -. Fused.
Vol. 2, 365-3 72, August 1991

cell Growth

Cell Adhesion Molecule Uvomorulin Breast Cancer Cell Lines: Relationship and Invasive Capacities

Connie L Sommers, Erik W. Thompson, Rolf Kemlen, Edward P. Gelmann, and Stephen W. Byers’

Jeffrey

A. Torn,

Vincent T. Lombardi

Cancer Research Center [C. L. S., E. W. 1., J. A. T., E. P. G., S. W. B.] and Department ofAnatomy and Cell Biology [C. L. S., E. W. T., E. P. G., S. W. B.], Georgetown University, Washington, DC 20007, and Max-Planck-Institut f#{252}r Immunobiologie, Stubeweg 51, D-7800 Freiberg, Federal Republic of Germany [R. K.]

Abstract adhesion in carcinoma cells may step in the acquisition of an invasive, phenotype. We have examined the

be an

Expression in Human to Morphology

carcinoma cells. Metastasis represents a critical event in the course of tumor progression, usually leading to tumor lethality in cancer patients. Invasion and metastasis of tumor cells into their surrounding connective tissue and to distant sites is a multistep process. Carcinoma cells must first detach from neighboring tumor cells, invade through the basement membrane, and migrate into surrounding tissue. The invading cells may then enter the vascular or lymphatic circulation. Finally, the cells may then attach to the wall of a capillary at a second site and pass

Loss of cell-cell

& Differentiation

through

the

basement

membrane

into

a target

or-

fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratinexpressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matnigel and measured the ability of the cells to traverse a Matnigel-coated filter as in vitro models for detachment of carcinoma cells

gan, where growth resumes. CAMs may play a role both in the detachment of tumor cells from a primary tumor and in reimplantation at a distant site. Other factors that are likely to be involved in invasion and metastasis are collagenases and other proteases, motility factors, and substrate adhesion molecules (1). In this study, we examine the expression of the CAM uvomorulin in breast cancer cell lines and propose that loss of uvomorulinmediated cell-cell adhesion may be one event required for initial local invasion potentially leading to metastasis in these cells. Uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) is a 120 kilodalton cell surface glycoprotein involved in calcium-dependent cell-cell adhesion (see Refs. 2-5 for reviews). It is expressed early in mouse development and

from

is involved

important metastatic expression of the epithelial-specific molecule uvomorulin (E-cadhenin, L-CAM)

in human

neighboring

breast

cells

cancer

and

cell adhesion cell-CAM 120/80, cell

invasion

lines.

We

through

find

that

basement

morphology

in Matnigel.

responsible Matnigel

We

show

that

uvomorulin

for the fused colony morphology

since

treatment

is

in

of uvomorulin-positive

MCF-7

cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness

in these

cells,

as measured

by their

ability

to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulinnegative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one progression

phenotype. Introduction may

Received

12/26/90. requests for reprints should be addressed, and Cell Biology, Georgetown University,

To whom of Anatomy I

20007.

play a role

in the invasion

and metastasis

heart

Compaction

of

preimplantation

em-

of

(6).

The

cell

lines

which

appeared

fibroblast-like

ex-

pressed vimentin, whereas the epithelioid cell lines expressed only keratins. There is increasing evidence that vimentin may be a marker for poor prognosis in breast cancer(8-10), and vimentin-expressing breast cancer cell lines are more invasive as measured by in vitro assays.3 Here, we find that fibroblastoid, invasive, vimentin-expressing breast cancer cell lines do not express uvomo-

2

at the Department Washington, DC

tissue

To investigate a possible correlation between CAM expression and invasiveness in human breast cancer, we analyzed 1 7 human breast cancer cell lines for the expression of uvomorulin. We have previously described the morphology of these cell lines, which correlated with expression of the intermediate filament protein vimentin (7).

of many changes involved in the of a carcinoma cell to an invasive

CAMs2

in the

bryos. In the adult, it is expressed specifically in epithelial cells. Disruption of uvomorulin function in nontumorigenic MDCK epithelial cells by specific antibody treatment resulted in a change from a cobblestone, epithelioid appearance to a more elongated, fibroblastic morphology when the cells were plated on plastic. A small percentage of the antibody-treated cells also acquired the ability to invade collagen gels and embryonal

membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate

The abbreviations

used are: CAM,

Madin-Darby canine kidney cells; Iscove’s minimal essential medium; 3

E. W.

Thompson

et a!.,

submitted

cell adhesion

molecule;

cDNA, complementary SSC, standard saline for

publication.

MDCK DNA; citrate.

cells, IMEM,

365

366

Uvomorulin

Expression

in Breast Cancer

Cell Lines

rulin. Another subset of keratin-expressing (vimentinnegative), less invasive cell lines also does not express uvomorulin. In general, these cell lines are not more invasive than other vimentin-negative, uvomorulin-positive

assay

breast cancer cell lines, as measured by an in vitro in which cells must penetrate a basement mem-

brane matrix-coated, porous, polycarbonate filter. These results indicate that although loss of uvomorulin-mediated cell-cell adhesion may facilitate invasion and metastasis, absence of uvomorulin expression does not, by itself, in vitro

lead to an invasive phenotype assay described here.

as detected

by the

embryos (13, 14). As illustrated in Fig. 4, b and of MCF-7 cells with either the gp84 or the DECMA-i antibody caused the cells to detach from each other in the Matrigel outgrowth assay, giving a morphology similar to that seen in uvomorulin-negative, vimentinnegative cells. This effect was seen whether the cells were treated with antibody at the time of plating or whether antibody was added the day after plating. Cells began to detach within 4 h after antibody treatment. In addition, this effect was reversible since, after removal ofgp84 and DECMA-1 antibodies by changing the media, the cells exhibited the fused, uvomorulin-positive pheplantation

e, treatment

notype Results

Seventeen uvomorulin

breast

cancer

expression

cell by

lines

were

analyzed

immunofluorescence

for micro-

and Northern blot analysis. Cell lines positive for uvomorulin expression, such as MCF-7, T47D, and MDAMB-i 75-7 (Fig. 1 a-c) showed bright staining at cell-cell borders by immunofluorescenCe microscopy similar to that previously described for MCF-7 (1 1) and other epithelial cells such as MDCK cells (6). Negative cell lines showed no specific cell-cell border staining (Fig. 1, d-f). A summary of uvomorulin expression in the 1 7 cell lines examined is presented in Table 1. scopy

,

Northern

blot

analysis

was

performed

to

investigate

whether uvomorulin mRNA was present in the breast cancer cell lines that were negative by immunofluorescence microscopy. As shown in Fig. 2a, a band of approximately 5 kilobases was seen in three breast cancer cell lines that were positive by immunofluorescence microscopy (MCF-7, BT474, and MDA-MB-361 No uvomorulin mRNA could be detected in cell lines MDA-MB231 and HS578T, even when MDA-MB-231 polyadenylated RNA was used (Fig. 2b). To show the presence of intact RNA in the MDA-MB-231 and HS578T lanes in Fig. 2a, the blot was subsequently hybridized to a vimentin cDNA probe. Both MDA-MB-231 and HS578T express the intermediate filament protein vimentin as previously described (7), whereas MCF-7, BT474, and MDA-MB361 do not express vimentin. It is interesting to note that the MCF-7ADR line, selected for resistance to adriamycin (12), has lost expression of uvomorulin and acquired vimentin expression. We investigated the morphology of the breast cancer cell lines plated in the reconstituted basement membrane matrix Matrigel, since potentially invasive carcinoma cells are in contact with basement membrane in vivo. The uvomorulin-positive, vimentin-negative cells form large, well-circumscribed colonies of cells in Matrigel in which individual cell borders are not distinguishable, and cells appear fused (Fig. 3, a and b). Uvomorulin-negative, vimentin-negative cells form clusters of spherical cells in Matrigel in which cell borders are easily distinguishable (Fig. 3, c and d). The uvomorulin-negative, vimentinpositive cells have a stellate morphology in Matrigel (Fig. 3, e and 1). To investigate whether the phenotype of fused cobnies versus colonies composed of separated cells was indeed dependent on uvomorulin expression, uvomo).

rulin-positive

MCF-7

polyclonal antiserum monoclonal antibody been shown to block

cells

were

treated

with

gp84

rabbit

to uvomorulin and DECMA-1 rat to uvomorubin, both of which have cell-cell interaction in mouse preim-

seen

in

untreated

cells

(Fig.

4c).

As

negative

cells were treated with similar concentrations of normal rabbit serum (Fig. 4d) or a rat monoclonal antibody to CD44/hyaluronate receptor (15, 16) (Fig. 41). CD44/hyaluronate receptor is an actin-assoCiated cell adhesion molecule involved in lymphocyte homing and in interactions with extracellular hyaluronate (16). After treatment with normal rabbit serum or CD44/ hyaluronate receptor antibody, MCF-7 colonies in Matrigel remained fused, demonstrating the specific involvement of uvomorulin in this phenotype. As an in vitro measure of invasive capacities, we tested the abilities of some of the breast cancer cell lines to cross a Matrigel-coated, porous, polycarbonate filter in a modified Boyden chamber assay. Three uvomorulin-positive, vimentin-negative cell lines were tested, as were two uvomorulin-negative, vimentin-negative and two uvomorulin-negative, vimentin-positive lines (Table 2). The vimentin-negative lines were less invasive than the vimentin-positive lines, as previously reported.3 Of the vi mentin-negative cell lines, the uvomorulin-negative lines CAMA1 and SK-BR-3 were not more invasive than uvomorulin-positive cells as measured by this assay. Since these cell lines were not more invasive, we also tested the effect on invasiveness of disrupting uvomorubin function in uvomorulin-positive MCF-7 cells. In a separate experiment, MCF-7 cells were treated with the same concentration of DECMA-1 antibody to uvomorulin that produced an effect in the Matrigel outgrowth assay and were assayed in the modified Boyden chamber chemoinvasion assay. The antibody did not increase invasiveness in MCF-7 cells; in fact, invasiveness was somewhat decreased. A similar effect was observed in the uvomorulin-positive cell lines T47D and BT474 (data not shown). As can be observed by Comparing Tables 2 and 3, some interassay variation was observed; therefore, the cells listed in Table 2 were assayed at one time, as were controls,

MCF-7

the cells

listed

in Table

3. Treatment

of MCF-7

cells

with

the anti-CD44/hyaluronate receptor antibody had no effect on invasion (Table 3). Therefore, in this assay system, loss of uvomorulin function did not, by itself, lead to increased invasiveness. Discussion

A significant suggested by ogy to neural gene deleted may represent

role for CAMs in malignant progression is several recent studies. A gene with homolCAM, DCC, is a putative tumor suppressor in 70% of coborectal cancers (1 7). This gene a novel CAM whose loss contributes to

malignant

progression

tion of resulted

B-lymphoblastoid in a specific

in coborectal

cancer.

cells by the down-regulation

Transforma-

myc oncogene of the integrin

Cell

Growth

& Differentiation

367

d

e

fig.

1.

“Materials 231;t,

Uvomorulin expression and Methods” usingthe BT549.

as detected by rabbit polyclonal

immunofluorescence antibody against

microscopy. uvomorulin,

Immunofluorescence gp84. a, MCF-7; h, T47D;

microscopy c,

was

MDA-MB-175-7;

performed

as described e, MDA-MB-

d, HS578T;

in

368

Uvomorulin

Tab!e

Expression

1

in Breast

Uvomorulin

Cancer

expression

Cell

in human

Lines

breast

can cer cell

Uvomorulin”

MDA-MB-361 MDA-MB-175-7 BT483

mRNA

IF

+++ +++ +++

+ + +

Fused

+ + + +

Fused Fused Fused Fused

-

++ ++ ++ ++ +

-

-

-

-

-

-

-

-

-

-

-

-

Spherical Spherical Spherical Spherical

+ 4+ + +

-

-

Stellate

-

-

Stellate

-

-

-

-

-

T47D ZR-75-B BT474 MCF-7 MDA-MB-468

-

SK-BR-3 MDA-MB-134 CAMA1 MDA-MB-453 MDA-MB-231 MDA-MB-436

BT549

,

HS578T MDF-7ADR Vimentin mRNA

b

Uvomorulin

Morphology . in Matrigel

Vimentin

Cell line

expression

as detected

10 days,

then

Fused

Stellate Stellate SteIIate

-

by Northern

microscopy

Fused Fused

-

-

as detected

or immunofluorescence Methods. ( Spherical for about

lines

blot

by

analysis.

Northern

(IF),

blot

as described

analysis

(mRNA)

in ‘Materials

and

stellate.

mentin negative, (2) uvomorulin negative/vimentin negative, and (3) uvomorulin negative/vimentin positive. Although loss of uvomorulin-mediated cell-cell adhesion may allow cells to detach from one another, an event which is likely to be required for local invasion, some breast cancer cell lines that did not express uvomorulin were not highly invasive, as measured by the in vitro assay described here. Clearly, additional changes other than uvomorulin loss are involved in the progression to a more invasive phenotype. One important event in the acquisition of invasive properties seems to involve a change in morphology accompanied by the onset of vimentin expression as represented by the uvomorulinpositive/vimentin-positive breast cancer cell lines, which were highly invasive. Other changes that are necessarily involved in invasion through basement membrane are increases in collagenase and other protease activities involved in breaking down basement membrane molecubes (19). This progression may be analogous to epithelial-mesenchymal transitions in embryonic development, in which keratin-expressing, uvomorulin-positive epithehal cells lose their surface polarity and give rise to migrating, uvomorulin-negative, vimentin-expressing mesenchymal cells. These cells can later reorganize into

epithelia CAM lymphocyte decrease in cell-cell

function-associated antigen 1 and a adhesion (18). We believe that these

studies and others point to the involvement of decreased homotypic cell-cell adhesion in tumor progression. Loss of cell-cell adhesion may occur by loss of expression, as

observed

in our

study,

or by reduction

unstable expression, or mutation. We have characterized breast cancer have varying degrees of invasive potential expression.

the

We

following

have

observed

phenotypes:

three

or differentiate

tives (20). transition,

into

connective

If primary carcinoma cells their ability to metastasize

enhanced. A correlation

between

can

uvomorulin

tissue undergo could be

derivasuch a greatly

expression

and

of expression,

metastatic ability has been observed in the murine ovarian carcinoma cell line OV2944 (2 1 ). A subline of OV2944 that was highly metastatic in nude mice had variable but low levels of uvomorulin compared to two weakly met-

cell lines that for uvomorulin

astatic

groups

of cells

(1) uvomorulin

with

positive/vi-

a

sublines,

which

consistently

expressed

high levels

of uvomorulin. These data also suggest that a loss or instability of uvomorulin expression correlates with increased metastatic ability. A more direct test of the

b H

H

ci,

(0

(Y)

c’_)

N

th

co

I-