Download E-Cadherin Expression in Human Breast Cancer

ICANCER RESEARCH56. 4063-4070. September 1. 19961
E-Cadherin Expression in Human Breast Cancer Cells Suppresses the Development
of Osteolytic Bone Metastases in an Experimental Metastasis Model'
Gabriel Mbalaviele, Cohn R. Dunstan, Akira Sasaki, Paul J. Williams, Gregory R. Mundy, and Toshiyuki Yoneda2
Unirersitv of Texas Health Science Center, Department
of Medicine. Division of Endocrinology
and Metabolism.
San Antonio, Texas 78284-7877 1G. M., C. R. D.. P. J. W..
G. R.M., T. Y.J,andDepartmentof Oral andMaxillofacialSurgeryII, OkayamaUniversitySchoolof Dentistry.Okayama700,JapanIA. 5.1
ABSTRACT
The molecular mechanisms by which human cancer cells spread to
bone are largely unexplored. The process likely involves cell adhesion
molecules (CAMs) that are responsible for homophilic and heterophilic
cell-cell interactions. One relevant CAM may be the calcium-dependent
transmembrane glycoprotein E-cadherin. To investigate the involvement
of E-cadherin in breast cancer metastasis to bone, we used an in vivo
model
in which
osteolytic
bone
metastases
preferentially
occur
after
ever, they do not show whether E-cadherin expression affects the
behavior of cancer cells per se during invasion and metastasis.
Treatment of noninvasive epithelial Madin-Darby canine kidney
cells with monoclonal antibodies to E-cadherin in vitro rendered these
cells more invasive (12). Expression of the E-cadherin gene in highly
invasive cancer cells dramatically
suppressed
conversely,
introduction
of E-cadherin-specific
their invasiveness
and,
antisense RNA ren
dered noninvasive epithelial cells invasive in vitro ( 13). E-cadherin
in
jections ofcancer cells directly into the arterial circulation through the left
ventricle of the hearts of nude mice. We have found that E-cadherin
negative human breast cancer cells MDA-MB-231 (MDA-231) develop
radiographically detectable multiple osteolytic bone metastases and ca
chexia in this model. However, MDA-231 breast cancer cells that were
transfected with E-cadherin cDNA showed a dramatically impaired Ca
pacity to form osteolytic metastases and induce cachexia. Histological and
histomorphometrical analyses of bones of mice bearing mock-transfected
MDA-231 revealed aggressive metastatic tumor, whereas metastatic tu
mor burden was significantly decreased in the bones of mice bearing
was strongly expressed in breast cancer cells with reduced invasive
ness, whereas relatively
low levels of E-cadherin
were detected in
E-cadherin-expressing
likely that the expression
of E-cadherin
inhibits detachment
and
invasion of cancer cells at the primary sites before the step of intrav
asation. Conversely,
however, it is also possible that, subsequent
to
intravasation,
E-cadherin
may rather facilitate the arrest of cancer
fected
MDA.231.
MDA-231
breast
cancer
Nude
mice
cells survived
bearing
longer
E-cadherin-trans
than
mice
bearing
mock-transfected MDA-231 breast cancer cells. Anchorage-dependent
and -independent growth in culture and tumor enlargement in the mam
mary fat pad of nude mice were unchanged between mock-transfected and
E-cadherin-expressing MDA-231, suggesting that these differences in met
astatic behavior
are not due to an impairment
highly invasive human breast cancer cells (14). These experimental
results show the inhibitory effects of E-cadherin
on the local inva
siveness of cancer cells and are consistent
with the notion that E
cadherin is an “invasionsuppressor gene―( 13). However, they do not
demonstrate whether E-cadherin expression also suppresses cancer
metastasis
organs.
Because
multiple
sequential
steps
are
cells at metastatic sites or coaggregation of cancer cells with platelets
in capillaries,
of cell growth and tumor
igenicity. Our results show the suppressive effects of E-cadherin expres
sion on bone metastasis by circulating breast cancer cells and suggest that
the modulation of expression of this CAM may reduce the destructive
effects of breast cancer cells on bone.
to distant
required for cancer cells to advance to achieve distant metastasis, it is
resulting
in a promotion
of metastasis.
Determining
whether this is the case seems to be important to our understanding of
the role of E-cadherin
expression
in distant
metastasis
of cancer.
We have recently developed an experimental metastasis model in
nude mice in which inoculation of cancer cells into arterial circulation
through the left cardiac ventricle selectively results in osteolytic bone
metastases
INTRODUCTION
(15), based
on the method
described
by Arguello
et a!.
(16). Using this model of bone metastasis, we examined the role of
E-cadherin in bone metastasis of human breast cancer because breast
E-cadherin (uvomorulin) is a Mr 120,000 transmembrane glycopro
tein involved in calcium-dependent cell-cell adhesion. Cell-cell adhe
cancer
frequently
colonizes
bone (17,
18). The E-cadherin-negative
homotypic or heterotypic cell aggregation, which is likely important
in controlling embryogenesis, morphogenesis, and tissue-specific im
munity (1 , 2). Recently, evidence has accumulated that E-cadherin
human breast cancer cell line MDA-23 1â€w̃as transfected with mouse
E-cadherin cDNA and studied for the capacity of these cells to
develop osteolytic bone metastases. Our data provide in vivo experi
mental evidence that functional E-cadherin expression in MDA-23 I
also plays a key role in cancer invasion and metastasis.
Immunohis
tochemical
examination
of clinical tumor specimens of the head and
breast cancer cells caused a marked decrease
metastases
even after these cells were directly
neck (3), prostate (4), stomach (5), kidney (6), and other organs (7)
has shown that E-cadherin expression is decreased in invasive and
metastatic cancers and that decreased E-cadherin expression is asso
blood stream. The results suggest that E-cadherin might have sup
pressive effects not only on the local invasiveness of cancer cells at
primary sites but also on the metastasis of circulating cancer cells to
distant organs such as bone.
sion mediated
ciated
by E-cadherin
with poor prognosis
differentiated
cadherin
clinical
is homophilic
or heterophilic,
(6, 8). It has also been shown
causing
that poorly
metastatic breast cancers express lower levels of E
than
studies
do well-differentiated
suggest
breast
that E-cadherin
cancers
expression
(9—11). These
is inversely
MATERIALS
corre
lated with the invasiveness and metastatic potential of cancer. How
Received 4/22/96; accepted 7/2/96.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
by NIH Grants
P0 1-CA40035,
CA58 I 83 (Specialized
Program
whom
requests
for
reprints
breast
was cultured
be
addressed,
at
Department
of
cell line MDA-23l
(Ref.
19; kindly
provided
by Dr. C.
in DMEM
(Hazleton
Biologics,
Inc., Lenexa,
KS) supplemented
with 10% FCS (Hyclone Laboratories, Logan, UT) and 1% penicillin-strepto
solution
atmosphere
should
cancer
Kent Osborne, University of Texas Health Science Center, San Antonio, TX)
mycin
of Re
search Excellence), and CA63628.
2 To
AND METHODS
MDA-231: A Human Breast Cancer Cell Line. The E-cadherin-negative
human
I Supported
in osteolytic
bone
introduced
into the
(Life Technologies,
Inc., Grand
Island,
NY) in a humidified
of 5% CO2 in air.
Medicine/
Endocrinology, University of Texas Health Science Center. 7703 Floyd Curl Drive, San
Antonio, TX 78284-7877. Phone: (210) 567-4900; Fax: (210) 567-6693; E-mail:
[email protected]
3 The abbreviations
used are: MDA-23
I, MDA-MB-23
I human breast cancer cell line:
MMP, matrix metalloproteinase: lBS. Iris-buffered saline.
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E-CADHERIN IN BREAST CANCER METASTASIS TO BONE
and transferred to polyvinylidene difluoride membranes (Immobilon-P; Milli
Transfection of Mouse E-Cadherin eDNA. MDA-231 (1 X l0@)cells
grown in DMEM supplemented with 10% FCS were cotransfected with 20 @xg
of full-length mouse E-cadherin cDNA (in pBATEM2 plasmid driven under
the control ofthe chicken 13-actinpromoter; Ref. 20) and 1 @xg
ofpSV2neo (21)
for neomycin resistance by calcium-phosphate coprecipitation method (22). As
a control, cells were also transfected with 2 @xg
of pSV2neo. The E-cadherin
expression plasmid, pBATEM2, was a generous gift of Dr. M. Takeichi
(Kyoto University, Kyoto, Japan). The neomycin expression vector, pSV2neo,
was purchased from Invitrogen (San Diego, CA). After 24 h of incubation,
cells were fed with fresh medium, grown for an additional 24 h, split and
seeded into 100-mm dishes, and cultured in the presence of 470 @xg/ml
of
antibiotic
G418
(Life Technologies,
Inc.).
G4l8-resistant
colonies
pore, Bedford,
20%
Staining
of E-Cadherin.
(pH
8.0)].
The
buffer
membranes
[20 mM Tris,
were
150 mist glycine,
incubated
overnight
and
with
blocking buffer [5% BSA in 50 mrviTris, 150 mrviNaCI, and 10 mMCaCI2]and
then with monoclonal antibodies to mouse E-cadherin (Transduction Labora
tories, Lexington, KY) diluted I :5000 in the same buffer for 2 h, washed once
with TBS + 0.05% Tween 20 (TBST), then twice with TBS, and incubated
with horseradish peroxidase-conjugated goat anti-mouse IgG (Capel, Durham,
NC; diluted 1:1500 in TBS containing 5% nonfat dry milk) for 1 h. After the
membranes were washed three times with TBST and then three times with
TBS, the signal was visualized with an enhanced chemiluminescence detection
system (DuPont NEN, Boston, MA).
Colony Formation Assay. Anchorage-independentgrowth of MDA-231
were iso
lated and cloned by limiting dilution twice.
Immunofluorescent
MA) in transblotting
methanol
cells was determined
Cells were fixed with a
as described previously (23). One ml of agarose (Sea
fresh solution of 3.7% (w/v) formaldehyde in PBS at room temperature for 30
mm, incubated with the primary antibodies (ECCD-2; Zymed Laboratories,
Inc., San Francisco, CA) at a 1:200dilution in PBS containing 1%rabbit serum
at 37°Cfor 1.5 h in humidified atmosphere, washed three times with PBS, and
incubated with 1:80-diluted affinity-purified rabbit anti-rat IgG conjugated
Plaque; FMC Bioproducts, Rockland, ME) at 0.4% (wlv) in DMEM supple
mented with 10% FCS containing 300 cells was overlaid on a 1-mi bottom
layer of 0.6% agarose in the same culture medium in 35-mm tissue culture
plates with a grid at the bottom (Sarstedt, Newton, NC). Plates were incubated
with fluorescein 5-isothiocyanate
100 ,xm in diameter were manually counted under an inverted microscope.
Cell Growth in Monolayer. Cells (1 x l05/well, 6-well plate; Falcon;
Becton Dickinson Labware, Lincoln Park, NJ) were cultured in DMEM
washing
with PBS,
examined
under
cells
for 14 days in a humidified CO2 incubator at 37°C,and colonies larger than
(Sigma Chemical Co., St. Louis, MO). After
were
mounted
a fluorescence
in PBS-glycerol
microscope
(Nikon
solution
(1:1)
Inc. Instrument
and
Division,
GardenCity,
NY).
Immunoblotting. Cells were lysed in lysis buffer [20 mMTris (pH 8.0), 2
mM CaCl2,
150 mrsi NaC1, 1% NP4O, 0.1%
mM leupeptin,
SDS, and protease
1 mM phenylmethylsulfonylfluoride,
inhibitors
and 1% aprotinin)]
supplemented
with
10% FCS. After the indicated
time of culture,
cells were
trypsinized and counted on a hemocytometer under an inverted microscope.
Intracardiac
(20
Injections
of MDA-231
Cells in Nude Mice. Intracardiac
injection of MDA-23l cells was performed according to the technique de
at 4°C
and centrifuged at 10,000 X g for 10 ruin at 4°C,and protein amounts were
scribed
determined
medium 24 h before intracardiac injections. Cells (1 X l0@)were suspended in
using the Bio-Rad
DC protein
assay (Bio-Rad
Laboratories,
Her
cules, CA). The lysates were boiled for 5 mm, separated on 7.5% SDS-PAGE,
I
previously
0.1 ml of PBS
2
3
(24).
Subconfluent
and injected
4
using
MDA-23l
a 27-gauge
5
6
cells
needle
were
fed with
fresh
into the left heart
7
kDa
-195
—
-@Ti
@
:@
-112
-84
-63
Fig. 1. E-cadherin expression in MDA-231 cells
in Western blotting (top) and immunofluorescence
(bottom). Top: Lane 1, MCF-7 as positive control;
Lane
2, Neo-MDA-231;
Lanes
3-7,
231 subclones. Bottom, phase-contrast
C
A
E-cad-MDA
microscopy
(A and C) and immunofluorescence (B and D).
Neo-MDA-23l showed no E-cadherin expression
(top, Lane 2; bottom, A and B). In contrast, E-cad
MDA-231 subclones strongly expressed E-cadherin
[top, Lanes 3-7; bottom, C and D (subclone 4)].
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E-CADHERIN IN BREAST CANCER METASTASIS TO BONE
Determination
A
of the Number
and Area
of Bone Metastases
The num
ber of osteolytic bone metastases was enumerated on radiographs as described
Cell Numberx105
(15, 24). Animals were anesthetized deeply, placed in prone and lateral
positions against the films (22 X 27 cm; X-OMAT AR; KOdak, Rochester,
NY), and exposed to an X-ray at 35 kV for 6 s using a Faxitron radiographic
15
0 Neo-MDA-231
inspection unit (Model 43855A; Faxitron X-ray Corporation, Buffalo Grove,
IL). Films were developed using a KOdak RP X-OMAT processor (Model
M6b). All of the radiographs of bones in nude mice were evaluated extensively
and carefully by three individuals (including one radiologist) who had no
]E-cad-MDA-231
knowledge
of the experimental
protocols.
On radiographs,
the number
and area
of osteolytic metastatic foci as small as 0.5 mm in diameter, which were
recognized as demarcated radiolucent lesions in the bone, were quantitatively
assessed using a computer-assisted Jandel Video Analysis (JAVA) image
analysis system (Jandel Scientific, Corte Madera, CA).
Tumorigenicity of MDA-231 Cells in Nude Mice. Cells (2 X 106)were
suspended
0
24
0
72
48
in 0.2 ml of the mixture
(1:1) of PBS and Matrigel
(Collaborative
Research,
Bedford,
MA)andinoculated
intotherightthoracic
mammary
fat
pad of female nude mice using 23-gauge needles as described previously (26).
Four weeks later, the tumors formed were excised and their weights were
CultureTime(h)
determined.
B
Histological,
Histomorphometrical,
and Stereological
Examination
of
Metastatic Cancer Burden. The details of these methods were described
Colony/well
previously
(24). In brief, forelimbs
and hind limbs from animals in each
treatment group were fixed with 10% formalin in PBS (pH 7.2) and decalcified
in 14% EDTA solution for 2—3weeks. Paraffin sections were
conventional
methods. The area of metastatic cancer infiltrations
T@
made using
was meas
ured in the distal femoral and proximal tibial metaphyses of both limbs in
longitudinal decalcified sections stained by H&E. A level showing the full
width of the growth plate and shaft was selected for measurement. All
metastatic cancer cells from the joint surface to a point 4 mm down the shaft
were measured in both the bone and where it had expanded into the surround
200
1@
ing soft tissues.
Metastases
in the epiphyses
were also included.
Statistical Analysis. All data were analyzed by Mann-Whitney test for
nonparametric
—
Neo-MDA-231
C
mals
was
samples. The statistical difference of survival rate of the ani
analyzed
by
generalized
Wilcoxon
test.
All
data
were
presented
as
the mean ±SE.
E-Cad-MDA-231
RESULTS
Tumor Weight (9)
2.0
Transfection of E-Cadherin cDNA into MDA-231 Cells. Subse
quent to transfection, G4l8 selection, and cloning by limiting dilution,
several subclones of MDA-23 1 breast cancer cells were obtained.
MDA-231 subclones transfected with E-cadherin cDNA were desig
nated E-cad-MDA-23l, and MDA-23l subclones transfected with
pSV2neo were designated neo-MDA-23 1 and used as controls in the
following experiments. E-cad-MDA-231 cells expressed E-cadherin
I
1',
Body weight (g)
Neo-MDA-231
E-Cad-MDA-231
@,
Fig. 2. A, growth curve of neo-MDA-23 I and E-cad-MDA-23 1 (subclones 4-7 of Fig.
1. top) in monolayer. Cells (1000/cm2) were plated and cultured for 24, 48, and 72 h. Cell
number was counted manually after trypsinization on a hemocytometer (n = 4). B,
anchorage-independent growth of neo-MDA-231 (0) and E-cad-MDA-23I subclone 4
(U).Threehundred
cellswereplatedin softagarin 35-mmtissueculturedishes,
andthe
number of colonies larger than 100 p@min diameter was counted microscopically after 14
days of culture (n = 6). C, growth of neo-MDA-23 1 (0) and E-cad-MDA-23 I subclone
4 (U) in nude mice. Two X 106 cells in the mixture of PBS and Matrigel (1:1) were
inoculated in the right thoracic mammary fat pad of4 week-old female nude mice. Tumors
formed were excised, and their weight was determined 4 weeks after inoculation as
described in the text. Each group had four animals. Values shown are mean ±SE.
*
*
20•
15•
.
10•
5
0
-
--.-
@
=_J._
I
.
.
.
ventricles of 4-week-old female BALB/c-nu/nu mice (Harlan Industries, Hous
ton, TX) under anesthesia with pentobarbital (0.05 mg/g). Animals were kept
.
. . .
.
.
.
in our animal
facilities
for 4-7 weeks as descnbed
(25). The weight
of animals
and excised tumors was determined by using a Lum@O@GramTM
(Ohaus Scale
Corporation, Florham Park, NJ).
Neo-MDA-231
6
--
7@
E.cad-MDA-231
Fig.3. Bodyweightof nudemicebearingneo-MDA-23
1andE-cad-MDA-23
1. Data
shown in Figs. 3, 4, 5, and 6 were obtained from the same experiments
and experiments
.
.
were repeated twice. Four E-cad-MDA-231 subclones (see Fig. I . top) were examined.
Valuesshownaremean±SE(n = 5 X 2 experiments).*, significantlydifferentfromthe
neo-MDA-231group(P < 0.01).
4065
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E-CADHERIN IN BREAST CANCER METASTASIS TO BONE
Neo-MDA-231
vitro and in vivo results convincingly
E-cad-MDA-231
I
@,
/
_1@
-@
trabecular
@
—
@@AI
‘L'5'@
E-cad-MDA-231
demonstrate
that transfection
of
E-cadherin cDNA did not impair the capacity of growth and tumor
igenicity of MDA-23 1 cells.
Development of Cachexia and Osteolytic Bone Metastases.
Nude mice injected with neo-MDA-23 1 cells into the left ventricle of
the heart showed profound cachexia (loss of muscle, fat, and body
weight; Fig. 3 and Fig. 4, top) and multiple osteolytic lesions in the
upper and lower extremities (Fig. 4, bottom and Fig. 5) 3—4weeks
after inoculation, as we reported previously (15, 24). On the other
hand, nude mice inoculated with E-cad-MDA-23l cells demonstrated
a marked impairment of cachexia (Fig. 3) and a significant decrease
in osteolytic bone metastases (Fig. 4, bottom and Fig. 5).
Histological View of Bones with Metastatic MDA-231 Cells.
Neo-MDA-23l cells almost completely destroyed the marrow archi
tecture, including the primary and secondary spongiosa of tibial bone,
and occupied the entire marrow cavity with frequent extension to the
surrounding soft tissue (Fig. 6). In contrast, E-cad-MDA-23 1 coloni
zation was relatively limited in the marrow cavity, and the majority of
bone and primary
and secondary
spongiosa
remained
intact
(Fig. 6). In both groups, enhanced osteoclastic bone resorption along
the endosteal surface of bone was observed adjacent to metastatic
MDA-231 cells (Fig. 6). The osteoclast number and bone resorption
surface area determined by histomorphometry were not different in
bones of E-cad-MDA-231- and neo-MDA-23l-bearing mice (data not
shown).
Histomorphometrical
Analysis of Metastatic Cancer Burden in
Bone. Consistent with the histological view, histomorphometrical
analysis of bones with neo-MDA-23 1 cells showed a larger metastatic
cancer burden than bones with E-cad-MDA-23 1 cells (Fig. 7).
Metastasis in Non-Bone Organs. As a characteristic feature of
this experimental metastasis model and by yet-unexplained mecha
Lesion number
10
4@I
p
.@
--
p
7.5
4'
5@
Fig. 4. Representative pictures (top) and radiographs (bottom) of nude mice bearing
neo (left)- and E-cad (right; subclone 4 of Fig. 1, top)-MDA-231 cancer. Arrows,
osteolytic bone metastases. Pictures and radiographs were taken 4 weeks after cell
inoculation. Note the differences in cachexia (loss of fat, muscle, and body weight) and
the number of osteolytic bone metastases between both groups.
2.5
*
1@
*
*
-I-
0
4
as determined by immunoblotting (Fig. 1, top) and immunofluores
cence (Fig. 1, bottom) and showed polygonal shape and increased
cell-cell contact (Fig. 1, bottom), suggesting that the E-cadherin
transfected was functional in MDA-231. Neo-MDA-231 cells showed
elongated shape and expressed undetectable levels of E-cadherin
(Fig.1).
Anchorage-dependent and -independent Growth and Tumori
genicity of E-cad-MDA-231
Cells. To determine whether E-cad
hem expression affects the growth of MDA-23l cells, the growth
curve of E-cad-MDA-23l cells was compared to that of neo-MDA
23 1 cells in monolayer
culture.
There was no difference
anchorage-independent
growth
4
3
7
+
2
*
*
I
in the growth
of neo-MDA-231
6
5
I
_
*
pattern between E-cad-MDA-23 1 and neo-MDA-23 1 cells (Fig. 2A).
Furthermore,
5
Le@ on area (mm @)
0
14
and
E-cad-MDA-23l cells assessed by colony formation in soft agar was
not significantly different (Fig. 2B).
More importantly, the weight of E-cad-MDA-23l tumors formed in
the mammary fat pad of nude mice 4 weeks after the inoculation was
not different from that of neo-MDA-23l tumors (Fig. 2C). These in
Neo-MDA-231
5
6
7
i
E-cad-MDA-231
Fig. 5. The number and area of osteolytic bone metastases in nude mice bearing neo
(EThandE-cad(U)-MDA-23
I cancer.
Thenumber
andareaofosteolytic
bonemetastases
were scored 4 weeks after cell inoculation on radiographs using quantitative image
analysis. Each group had five mice. Values shown are mean ± SE (si = 10). *.
significantly different from the neo-MDA-231 group (P < 0.005).
4066
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
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@
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E-CADHERIN IN BREAST CANCER METASTASIS TO BONE
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Fig. 6. Histological view of the proximal tibiae of nude mice bearing no cancer (A), neo-MDA-23l (B), and E-cad-MDA-23l (C; subclone 4 of Fig. 1, top). Almost all the primary
andsecondaryspongiosawerereplacedby metastaticbreastcancer(7) in miceinoculatedwithneo-MDA-231
(B)as comparedto theboneof non-tumor-bearing
normalmice(A).In
contrast, colonization of E-cad-MDA-23l (7) in the tibiae was localized, and the majority of the marrow cavity remained intact (C). with an appearance similar to that of
non-tumor-bearing normal mice. H&E staining, X40. Osteoclastic bone resorption along the endosteal surface of bone of nude mice bearing neo-MDA-23 1 (D) and E-cad-MDA-231
(5) at higher magnification.Osteoclasts(arrows) are presentbetween metastaticMDA-231 cells (7) and bone. H&E staining, X200.
nisms, very few metastases in other organs such as lungs, kidney,
brain (data not shown), and liver were detected in both groups
(Table 1). Therefore, it was not possible to statistically analyze the
died of cancer with osteolytic bone metastases (with analogous radio
graphical and histological views to those of neo-MDA-23 1-bearing
animals) 8 weeks after the inoculation. The results indicate that
effects
E-cad-MDA-23l
of E-cadherin
expression
on MDA-23l
metastasis
to non-bone
organs.
Survival of Animals. Nude mice inoculated with E-cad-MDA-23l
cells survived significantly longer than animals injected with nontransfected MDA-231 cells or neo-MDA-231 cells (Fig. 8). However,
it should be noted that all animals bearing E-cad-MDA-231 cells also
cells
did not lose
the capacity
to develop
bone
metastases, but that the capacity is impaired, presumably due to
E-cadherin expression.
Production of Bone-destroying Activity. To determine whether
E-cad-MDA-23l cells produce greater levels of osteolytic activities
than do neo-MDA-231 cells, serum-free culture supernatants were
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E-CADHERIN IN BREAST CANCER METASTASIS TO BONE
E-cadherin in E-cadherin-negative MDA-23l human breast cancer
cells suppresses metastasis to bone even after the cells have entered
the systemic circulation, diminishes loss of body weight, and prolongs
the life span of MDA-231 breast cancer-bearing animals. The results
provide experimental evidence that E-cadherin has a suppressive
action on distant metastasis of breast cancer cells to bone in vivo.
The mechanisms by which E-cadherin expression decreases osteo
lyric bone metastases in this animal model remain to be elucidated. In
this study, several subclones of MDA-23 1 cells that were transfected
with E-cadherin cDNA exhibited decreased formation of osteolytic
bone metastases, indicating that the results obtained here reflect the
phenotype of E-cadherin-expressing MDA-231 cells and are not due
to the clonal difference. Our data also demonstrate that E-cadherin
expression had no effects on the growth and tumorigenicity of MDA
231 cells. Frixen and Nagamine (31) have reported that E-cadherin
expression is associated with decreased secretion of proteolytic en
zymes. In our study, however, there was no change in MMP secretion
and bone-destroying activity in the absence or presence of E-cadherin
expression. Subsequent to entry into the systemic circulation, there are
still multiple diverse and complex steps including directional migra
tion, escape from host immune cell surveillance, and extravasation
Areaof Individualtumor
@
4 I
—r-
3'
2'
*
1'
0'
4
Neo-MDA-231
E-cad-MDA-231
Fig. 7. Histomorphometrical analysis of metastatic cancer burden of two ([1)- and
E-cad (U; subclone 4 of Fig. I, top)-MDA-231 in bone (proximal tibia). Values shown are
mean ±SE (n = 20 bones).*, significantlydifferentfromthe neo-MDA-231group
(P < 0.005).
through
harvested
and examined
for their
activity
in bone
resorption
assay
using 45Ca-labeled fetal rat long bones, as described (27). There was
no difference in the secretion of bone-destroying activity between
E-cad-MDA-231 and neo-MDA-23l cells (data not shown). More
over, we also examined MMP activity in the culture supernatants by
zymography.
However,
we did not observe
a significant
change
MMP secretion between E-cad-MDA-231 and neo-MDA-23l
(data not shown).
experimental
and clinicopathological
studies
have
is rare. Although
the mechanisms
underlying
in
cells
mdi
the preference
for bone to non-bone organs in this experimental metastasis model
remain unknown, this technique allows us to study the role of E
cadherin in the development of osteolytic bone metastases by human
breast cancer cells that are directly introduced into the arterial blood
stream. The data presented here clearly show that the expression of
Table 1 Dissemination
cells must progress
±—---E-cad-MDA-231
(n = 15)
(n = 15)
+++
+
to bone
++
±
they arrest
in bone.
demon
Clump
between
metastatic
cancer
cells and osteoclasts
that are modulated
by
either soluble factors or direct cell-cell contact or both in the bone
microenvironment
seems
to be necessary
for the development
of
osteolytic metastases. Along this line, it is noteworthy that our recent
data demonstrate that osteoclasts express E-cadherin (27). However,
of MDA-231 in the o
of nud mice
MDA-23 1 cells (1 X lOs) were inoculated into the left
cardiac ventricle. Four weeks after the inoc ulation, eache organwas
+),Muscle
metastasis-positive: (—),metastasis-negative; (±), occasionally metastasis-positive.rgans
adjacentBone
glandsBrainKidneyLungLiverOvaryNeo-MDA-23l
before
may involve the alteration of cell-cell adhesion
ing or clustering of MDA-23l cells may affect dispersion and migra
tion capacity in the circulation and reduce cancer cell passage through
bone capillary beds. Recent studies have described that much stronger
shear forces are required to dissociate the aggregates of E-cadherin
positive breast cancer cells than E-cadherin-negative breast cancer
cells (32) and that E-cadherin expression is inversely correlated with
cell motility (33). E-cadherin has been shown to mediate not only
homophilic but also heterophilic and heterotypic cell-cell contact (34)
and cell-substratum adhesion (35). Therefore, it is possible that the
interactions of MDA-23 1 cells with host cells and microenvironments
are modulated by E-cadherin expression, leading to decreased bone
metastases. To further study the role of E-cadherin in MDA-23l
metastasis to bone, we are currently examining chemotactic migra
tion, invasiveness, and adherence to various types of extracellular
matrix in neo- and E-cad-MDA-231 cells, using Boyden chamber
assay, Matrigel, and extracellular matrix-coated culture plates,
respectively.
There is a characteristic Step ifl bone metastasis that is not relevant
in metastasis to non-bone organs. Because the majority of bone
consists of calcified matrix and cancer cells do not apparently possess
an intrinsic capacity to destroy bone, the cooperative assistance of
osteoclasts, the only cells capable of resorbing bone, is required for
cancer cells to grow progressively in bone (17, 18). Thus, as a
mechanism specific for bone metastasis, a functional association
cated that E-cadherin impairs the local invasion of cancer cells into
surrounding tissue at the primary sites (3, 7, 11—14,28—30),which
may eventually result in the suppression of distant metastasis. How
ever, to date, little is known in regard to whether E-cadherin decreases
the metastasis of circulating cancer cells that have achieved intrava
sation. We attempted to address this question specifically for breast
cancer metastasis to bone because bone is the most common site of
metastasis in breast cancer (17, 18). To study this, we took advantage
of an experimental metastasis model in which the inoculation of
cancer cells into arterial circulation through the left heart ventricle of
female nude mice causes selective development of metastases in bone.
Nakai et a!. ( 15) and Sasaki et a!. (24) have already reported that, as
a characteristic feature of this model, cancer cells inoculated using this
technique preferentially metastasize to bone and develop osteolytic
lesions, whereas metastasis to other organs such as lung, liver, and
kidney
cancer
between MDA-23 I cells. E-cad-MDA-23
I cells in culture
strated changes in morphology
and increased cell-cell contact.
DISCUSSION
Previous
which
One mechanism
examined macroscopically
and microscopically.
Adrenal
——————
4068
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(
E-CADHERIN IN BREAST CANCER METASTASIS TO BONE
adhesion molecule E-cadherin is reduced or absent in high-grade prostate cancer.
Survival Rate (%)
Cancer Res., 52: 5104—5109,1992.
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Weeks
Fig. 8. Survival of nude mice bearing neo (broken line)- and E-cad (solid line: subclone
4 of Fig. I, top)-MDA-231 cancer. There were 14 mice in each group. *. significantly
E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship
different from the neo-MDA-231 group (P < 0.01).
12.
our results demonstrated that E-cadherin expression did not alter the
production of osteolytic activity and MMP secretion in MDA-23 I
cells in culture. Thus, it seems unlikely that E-cadherin expression
modulates the Secretion of soluble factors by metastatic MDA-23 I
breast cancer cells that stimulate osteoclastic bone destruction.
Enhanced cell-cell aggregation in E-cad-MDA-23 1 cells also mdi
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be explored.
In conclusion, we have found in the present study that the
expression of E-cadherin inhibits the metastasis of circulating
human breast cancer cells to bone in vivo. Inhibition of aj33
integrin by peptide antagonists or monoclonal antibodies has been
demonstrated to promote primary tumor regression by inhibiting
angiogenesis (42). In addition to integrins, the modulation of
expression of another cell adhesion molecule, E-cadherin, by ad
ministering agents that up-regulate E-cadherin expression may be
a potential therapeutic approach to inhibit the spread and destruc
tive effects of breast cancer in bone. Our results support the
feasibility of such a strategy.
I 3.
14.
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ACKNOWLEDGMENTS
We thank Drs. M. Takeichi and R. Kemler for providing us with plasmid
pBATEM2 and DECMA-l antibodies, respectively, and Thelma Barrios for
secretarial assistance.
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E-Cadherin Expression in Human Breast Cancer Cells
Suppresses the Development of Osteolytic Bone Metastases in
an Experimental Metastasis Model
Gabriel Mbalaviele, Colin R. Dunstan, Akira Sasaki, et al.
Cancer Res 1996;56:4063-4070.
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