Requirement for matrix metalloproteinase-9 (Gelatinase B) expression in metastasis by murine prostate carcinoma

Abstract

Although a number of effective therapies are available for localized prostate cancer, metastatic prostate cancer is difficult to treat and impossible to cure. Identification of the gene products that enable a prostatic carcinoma cell to metastasize should facilitate an understanding of the processes leading to metastasis. To characterize the contribution of matrix metalloproteinase-9 (MMP-9, gelatinase B or the 92-kd type IV gelatinase/collagenase) to the development of metastasis in prostate cancer, we reduced MMP-9 expression in metastatic murine prostatic carcinoma cells using a ribozyme. The ribozyme transfected cells had lower basal levels of MMP-9 as well as decreased levels after stimulation by transforming growth factor-beta or phorbol 12-myristate 13-acetate when compared with the parental cells or with control transfectants. The cells with down-regulated MMP-9 were unable to form lung colonies in the experimental metastasis assay, whereas the controls and parental cells readily formed metastases. All cell types readily formed tumors after injection and down-regulation of MMP-9 did not adversely affect the rate of tumor growth. Thus, MMP-9 expression is required for hematogenous metastasis in a murine prostate model system raising the possibility that it may play an equivalent role in human prostate cancer.

Full text

American
Journal
of
Pathology,
Vol.
152,
No.
2,
February
1998
Copyright
©)
American
Society
for
Investigative
Pathology
Requirement
for
Matrix
Metalloproteinase-9
(Gelatinase
B)
Expression
in
Metastasis
by
Murine
Prostate
Carcinoma
Geeta
Sehgal,*
Jin
Hua,*
Eric
J.
Bernhard,t
Inder
Sehgal,*
Timothy
C.
Thompson,f
and
Ruth
J.
Muschel*
From
the
Departments
of
Pathology
and
Laboratory
Medicine,*
and
Radiation
Oncology,t
University
of
Pennsylvania,
Philadelphia,
Pennsylvania
and
the
Department
of
Urology,*
Baylor
College
of
Medicine,
Houston,
Texas
Although
a
number
of
effective
therapies
are
available
for
localized
prostate
cancer,
metastatic
prostate
can-
cer
is
difficult
to
treat
and
impossible
to
cure.
Identi-
fication
of
the
gene
products
that
enable
a
prostatic
carcinoma
cell
to
metastasize
should
facilitate
an
un-
derstanding
of
the
processes
leading
to
metastasis.
To
characterize
the
contribution
of
matrix
metallopro-
teinase-9
(MMP-9,
gelatinase
B
or
the
92-kd
type
IV
gelatinase/collagenase)
to
the
development
of
metas-
tasis
in
prostate
cancer,
we
reduced
MMP-9
expres-
sion
in
metastatic
murine
prostatic
carcinoma
cells
using
a
ribozyme.
The
ribozyme
transfected
cells
had
lower
basal
levels
of
MMP-9
as
well
as
decreased
levels
after
stimulation
by
transforming
growth
factor-8
or
phorbol
12-myristate
13-acetate
when
compared
with
the
parental
cells
or
with
control
transfectants.
The
cells
with
down-regulated
MMP-9
were
unable
to
form
lung
colonies
in
the
experimental
metastasis
assay,
whereas
the
controls
and
parental
cells
readily
formed
metastases.
All
cell
types
readily
formed
tu-
mors
after
injection
and
down-regulation
of
MMP-9
did
not
adversely
affect
the
rate
of
tumor
growth.
Thus,
MMP-9
expression
is
required
for
hematoge-
nous
metastasis
in
a
murine
prostate
model
system
raising
the
possibility
that
it
may
play
an
equivalent
role
in
human
prostate
cancer.
(Am
J
Patbol
1998,
152:591-596)
In
individual
patients
it
is
currently
not
possible
to
predict
whether
metastasis
has
occurred
from
the
size
or
other
clinical
parameters
of
the
primary
tumor.
For
patients
with
prostatic
carcinoma,
the
determination
of
whether
metas-
tases
are
present
has
considerable
clinical
significance
as
prostate
cancer
can
often
be
effectively
treated
if
the
tumor
has
remained
confined
to
the
gland,
but
for
pa-
tients
with
metastatic
disease,
long
term
survival
is
rare.
Thus,
an
understanding
of
the
gene
products
that
are
required
for
metastasis
in
prostate
cancer
could
be
valu-
able
both
for
delineation
of
the
alterations
that
must
occur
in
the
cell
for
metastasis
to
occur
and
to
lead
to
the
identification
of
markers
that
might
predict
metastasis.
To
determine
some
of
the
factors
that
are
required
for
metastasis
by
prostate
carcinoma
cells,
we
turned
to
the
mouse
prostate
reconstitution
system
developed
by
Thompson
et
al1
in
which
neonatal
urogenital
sinus
tis-
sue,
the
fetal
precursor
of
the
adult
prostate,
was
ex-
planted,
infected
with
a
recombinant
retrovirus
that
ex-
presses
the
oncogenes
rasH
and
myc,
and
reimplanted
into
the
renal
capsule.
This
procedure
resulted
in
the
formation
of
tumors
that
histologically
were
virtually
iden-
tical
to
human
prostatic
carcinomas
and
that
displayed
characteristic
murine
prostate
markers.
These
tumors
were
generally
nonmetastatic,
but
when
male
p53
-/-
or
p53
+/-
mice
were
used
as
the
source
of
urogenital
sinus
tissue,
the
resultant
tumors
frequently
gave
rise
to
metastases
in
the
lung,
liver,
bone,
and
mesentery.2
Sehgal
et
al,3
using
cells
isolated
from
both
primary
tumors
and
metastases
generated
in
this
way,
found
that
the
105-kd
gelatinase
activity
characteristic
of
murine
matrix
metalloproteinase-9
(MMP-9)
or
murine
gelatinase
B
could
be
induced
by
transforming
growth
factor-,8
(TGF-3)
in
five
of
the
six
lines
isolated
from
metastatic
tumors
but
in
only
one
of
six
cell
cultures
isolated
from
the
primary
tumors.
These
data
correlating
MMP-9
produc-
tion
to
prostate
cancer
metastasis
suggested
that
MMP-9
expression
might
be
important
for
metastasis
in
this
sys-
tem
and
were
consistent
with
observations
linking
MMP-9
to
metastasis
in
other
systems.
MMP-9,
gelatinase
B,
or
the
92-kd
type
IV
gelatinase/
collagenase
is
one
member
of
a
family
of
matrix
metallo-
proteinases
(MMPs),
an
enzyme
family
defined
by
a
con-
served
catalytic
domain
that
requires
Zn2+
for
activity.4`6
MMP-9
is
capable
of
degrading
a
variety
of
substrates
including
molecules
found
in
the
extracellular
matrix
such
as
collagens
IV,
V,
elastin,
entactin,
as
well
as
casein,
the
precursor
of
tumor
necrosis
factor-a,
and
a
cell
surface
Supported
by
grants
from
the
National
Cancer
Institute
National
Institutes
of
Health,
CA-46830
(R.
J.
Muschel),
CA
50588,
and
CA
68814
(T.
C.
Thompson).
Accepted
for
publication
November
15,
1997.
Address
reprint
requests
to
Dr.
Ruth
J.
Muschel,
Room
269
John
Morgan
Building,
36
and
Hamilton
Walk,
University
of
Pennsylvania,
Phil-
adelphia,
PA
19104.
E-mail:
muschel@mail.med.upenn.edu.
591

592
Sehgal
et
al
AJP
February
1998,
Vol.
152,
No.
2
molecule,
galectin.7-9
MMP-9
is
secreted
in
an
inactive
form
of
molecular
mass
of
92
kd
in
humans
and
rats
but
of
105
kd
in
mice
because
of
a
16-amino
acid
insert.10'11
In
addition
to
the
widely
recognized
secreted
form
of
MMP-9,
Toth
et
al12
have
also
identified
a
cell
surface-
associated
form.
Successive
cleavages
of
the
N-terminal
region,
which
has
inhibitory
activity
against
the
enzyme
itself,
results
in
the
active
82-kd
form
and
sometimes
an
active
65-kd
form
of
the
human
enzyme.13
In
vitro
treat-
ment
of
pro-MMP-9
with
either
stromelysin,
MMP-2,
tissue
kallikerien,
matrilysin,
type
collagenase,
14-19
or
plas-
minogen
activator
generates
the
active
form,
but
the
physiological
activators
are
unknown.
The
tissue
inhibi-
tors
of
matrix
metalloproteinases,
TIMPs-1,
-2,
-3,
or
-4
can
inhibit
MMP-9
activity,
but
TIMP-1
has
a
higher
affin-
ity
for
MMP-9
than
does
TIMP-2,
and
MMP-9
is
frequently
isolated
in
a
complex
with
TIMP-1
.20-31
The
importance
of
MMP-9
in
metastasis
was
under-
scored
by
results
in
a
rat
sarcoma
model
system.
Bern-
hard
et
a132
noted
that
rat
embryo
fibroblasts
transformed
by
rasH
plus
myc
were
metastatic
and
secreted
MMP-9,
but
that
the
same
cells
transformed
by
ras
plus
adenovi-
rus
ElA
were
nonmetastatic
and
did
not
secrete
MMP-9.
Both
cell
types
were
equally
tumorigenic.
Introduction
of
ElA
expression
vectors
into
metastatic
transformed
rat
embryo
fibroblasts
inhibited
both
metastasis
and
MMP-9
expression.
ElA
will
also
block
metastasis
by
murine
mammary
cells.33
To
establish
the
role
of
MMP-9
in
this
system,
Bernhard
et
a134
introduced
an
expression
vector
for
MMP-9
into
the
nonmetastatic
rasH
plus
ElA
transfor-
mants.
This
led
to
metastasis
in
the
experimental
metas-
tasis
assay.
In
complementary
work,
Hua
and
Muschel35
showed
that
inhibition
of
MMP-9
expression
using
an
expression
vector
for
a
ribozyme
against
MMP-9
in
the
fibroblasts
that
were
transformed
by
rasH
plus
myc
blocked
experimental
metastasis
but
did
not
measurably
alter
tumorigenicity.
These
data
established
the
impor-
tance
of
MMP-9
in
metastasis
in
a
rat
sarcoma
model
system
as
well
as
its
independence
from
tumorigenicity
and
led
us
to
ask
whether
inhibition
of
MMP-9
expression
would
affect
metastasis
by
the
cell
lines
isolated
from
the
metastatic
tumors
generated
in
the
mouse
prostate
re-
constitution
system.
Materials
and
Methods
Plasmids
MMP-9
ribozyme
and
the
control
hammerhead
expres-
sion
vectors
were
constructed
by
subcloning
DNA
frag-
ments
coding
for
the
ribozyme
directed
against
MMP-9
or
for
a
hammerhead
structure
into
the
expression
vector
pRC/CMV
(Invitrogen,
San
Diego,
CA)
as
described
in
Hua
and
Muschel.35
Cell
Culture
Murine
prostate
carcinoma
cells,
the
metastatic
(148-
1,LMD)
and
primary
cells
(148-1,PA),
were
generated
as
described
in
Thompson
et
al.2
Cell
cultures
were
routinely
maintained
in
Dulbecco's
modified
Eagle's
me-
dium
supplemented
with
10%
heat-inactivated
fetal
bovine
serum,
penicillin,
and
streptomycin.
Metastatic
(148-1,LMD)
cells
were
transfected
with
30
,ug
of
pRC/
CMV,
pRC/CMV
with
the
MMP-9
ribozyme,
or
the
control
hammerhead
construct
by
the
calcium
phosphate
pre-
cipitation
method
as
described
previously
(Muschel
et
al,
1985).36
The
parental
cells
already
contain
one
copy
of
the
neomycin
resistance
gene
because
of
the
homolo-
gous
recombination
to
eliminate
p53.
The
parental
cells
retained
neomycin
resistance
at
600
to
700
,ug/ml.
Intro-
duction
of
the
ribozyme-expressing
plasmids
was
ac-
complished
by
selection
after
transfection
for
greatly
en-
hanced
resistance
to
geneticin.
Selection
was
with
geneticin
at
1.4
mg/ml.
Individual
colonies
were
isolated
using
cloning
cylinders.
Clones
AR
1.51
and
AR
1.52
were
isolated
after
transfection
of
148-1,LMD
with
the
expression
vector
for
the
MMP-9
ribozyme,
and
sub-
clones
AAS
1.22
and
AAS
1.74
were
isolated
after
trans-
fection
of
the
same
cells
with
the
expression
vector
for
the
hammerhead
control
ribozyme.
Zymography
Gelatin
substrate
gel
electrophoresis
was
accomplished
as
previously
described.32
To
prepare
conditioned
me-
dium,
cells
were
washed
with
serum-free
medium
and
resupplied
with
fresh
serum-free
media.
They
were
treated
with
TGF-f3
(80
pmol/L)
or
with
TPA
(50
ng/ml).
After
24
hours,
the
serum-free
conditioned
media
was
harvested,
adjusted
for
cell
number,
centrifuged
at
1500
x
g
to
remove
cellular
debris,
and
concentrated
to
650
,ul
(Centriprep-10
concentrator,
Millipore,
Burlington,
MA).
Concentrated
media
was
electrophoresed
under
nonreducing
conditions
and
without
heating
through
a
7%
sodium
dodecyl
sulfate-polyacrylamide
gel
electro-
phoresis
gel
containing
0.1%
porcine
gelatin
(Sigma,
St.
Louis,
MO).
Gels
were
washed
in
0.05
mol/L
Tris
(pH
7.4),
2%
Triton
X-100,
0.2
m
of
NaCI,
and
0.02%
NaN3.
Positive
controls
were
human
recombinant
MMP-9
generously
provided
by
R.
Fridman
(Wayne
State
University,
Detroit,
Ml)
or
conditioned
medium
from
2.10.10
cells
from
trans-
formed
rat
embryo
fibroblasts.37
Gels
were
stained
in
0.2%
Coomassie
blue
for
1
hour
and
destained
in
20%
(v/v)
methanol
and
10%
(v/v)
acetic
acid.
The
clear
bands
represent
gelatinase
activity.
Northern
Blotting
Total
cellular
RNA
(60
,ug)
or
polyadenylated
RNA
(5
,ug)
(direct
mRNA
mini
kit,
Qiagen,
Chatsworth,
CA)
was
elec-
trophoresed
in
a
formaldehyde/agarose
gel
and
trans-
ferred
to
a
Hybond-N+
membrane
(Amersham,
Arlington
Heights,
IL).
RNA
blot
hybridization
was
performed
with
a
32P-labeled
probe
derived
from
the
1.3-kb
EcoRI
insert
fragment
released
from
cDNA
clone
p8P2a37
or
from
the
ribosomal
protein
rPL32
as
an
internal
standard.3839
A
probe
that
hybridizes
to
the
ribozyme
was
derived
from
the
146-bp
insert
fragment
released
from
pRC/CMV

MMP-9
in
Prostate
Cancer
593
AJP
February
1998,
Vol.
152,
No.
2
0
Oo
C-
2
92
_
kDa
Primary
Metastatic
_.O
105
kDa
Figure
1.
Gelatinolytic
activity
is
present
in
conditioned
media
from
meta-
static
murine
prostate
carcinoma
cells.
Concentrated,
conditioned
medium
from
cells
derived
from
a
primary
prostate
carcinoma
148-1,PA
or
from
a
metastatic
tumor
148-1,LMD
were
subjected
to
gelatin
substrate
gel
electro-
phoresis.
The
cells
were
treated
for
24
hours
with
TGF-,3
or
with
TPA
as
indicated.
MMP-9
ribozyme
construct
by
digestion
with
Sacd
and
Apal.
Lung
Colonization,
Metastasis,
and
Tumorigenicity
Assays
Four-
to
six-week-old
NCr-nu/nu
mice
were
obtained
from
Taconic
Farms
(Germantown,
NY)
and
housed
asepti-
cally.
On
the
day
of
injection,
cells
were
washed
in
phos-
phate-buffered
saline,
trypsinized,
resuspended
in
se-
rum-free
Dulbecco's
modified
Eagle's
medium,
and
microscopically
examined
to
ensure
single
cell
suspen-
sions.
For
metastasis
studies,
mice
were
injected
in
the
lateral
tail
vein
with
5
x
104
single
cells/0.1
ml.
Animals
were
sacrificed
when
exhibiting
labored
breathing
or
at
4
weeks.
Lungs
were
fixed
in
10%
(w/v)
formalin.
A
dissect-
ing
microscope
was
used
to
count
the
lung
tumors
for
evidence
of
metastasis.
Tumorigenicity
was
assessed
after
injection
of
5
x
105
subcutaneously
in
the
flanks
of
4-
to
6-week-old
male
nu/nu
mice.
Six
tumors
were
mea-
sured
for
each
point.
The
tumor
volume
was
determined
by
measurement
of
the
tumor
in
three
dimensions
with
calipers
at
the
indicated
times.
Results
Cells
derived
either
from
a
primary
tumor
or
from
a
met-
astatic
tumor
generated
in
the
mouse
prostate
reconsti-
tution
system
were
evaluated
for
expression
of
MMP-9.3
Conditioned
medium
from
the
cells
isolated
from
a
pri-
mary
tumor
(148.1,PA)
showed
little
basal
expression
of
the
105-kd
gelatinase
activity
characteristic
of
murine
MMP-9,
and
there
was
no
increase
after
exposure
of
the
cells
to
either
TGF-,3
or
TPA
(Figure
1).
In
contrast,
the
conditioned
medium
from
the
cells
from
the
metastasis
(148.1,LMD)
contained
detectable
gelatinolytic
activity
at
105
kd
after
24
hours,
and
the
amount
of
gelatinase
released
increased
after
exposure
of
the
cells
to
either
TGF-,B
or
to
TPA.
The
conditioned
medium
from
the
pri-
mary
cells
had
a
high
level
of
gelatinolytic
activity
in
the
range
of
72
kd
likely
to
be
MMP-2,
but
this
activity
was
not
seen
in
the
medium
from
the
metastatic
cells.
Thus,
we
chose
to
use
the
148.1,LMD
prostatic
carcinoma
cells
isolated
from
a
metastasis
as
a
vehicle
to
test
whether
down-regulation
of
MMP-9
would
have
an
effect
on
met-
astatic
potential.
Hua
and
Muschel35
had
previously
shown
that
the
introduction
of
an
expression
vector
for
a
ribozyme
di-
rected
against
the
rat
MMP-9
could
down-regulate
MMP-9
expression
in
rat
cells.
This
ribozyme
consists
of
a
hammerhead-type
catalytic
site
flanked
by
15
bases
(8
on
the
5'
and
7
on
the
3'
side)
complementary
to
the
rat
MMP-9
mRNA.
Thirteen
of
these
15
bases
are
identical
between
rats
and
mice
suggesting
that
this
ribozyme
might
also
be
effective
against
murine
MMP-9.
After
transfection
of
this
vector
into
parental
148.1,LMD
cells,
11
transfected
clones
were
isolated;
six
showed
de-
creased
levels
of
released
MMP-9
gelatinase
activity.
Transfection
with
an
expression
vector
that
codes
for
a
ribozyme
hammerhead
structure
flanked
by
the
sense
MMP-9
sequence,
which
would
not
target
MMP-9,
was
used
as
a
control.
Of
the
six
clones
isolated
after
trans-
fection
with
the
control
ribozyme
expression
vector,
none
showed
decreased
levels
of
105-kd-released
gelatinase
activity
compared
with
the
parental
cells.
Two
clones
transfected
with
the
MMP-9
ribozyme
and
two
with
the
control
ribozyme,
were
selected
for
additional
analysis.
These
clones
were
shown
to
contain
the
plasmid
DNA
using
Southern
blotting
(data
not
shown).
Figure
2A
shows
that
mRNA
corresponding
to
the
ribozyme
could
be
detected
in
the
transfectants
but
not
in
the
parental
cells.
MMP-9
mRNA
could
not
be
detected
in
the
cells
transfected
with
the
MMP-9
ribozyme
under
the
same
conditions
in
which
MMP-9
mRNA
could
be
found
in
the
clones
transfected
with
the
control
ribozyme
(Figure
2B).
Increased
levels
of
MMP-9
mRNA
were
seen
in
the
con-
trol
cells
after
treatment
with
either
TGF-f
or
TPA,
but
MMP-9
mRNA
was
still
not
detected
in
either
of
the
two
clones
transfected
with
the
expression
vector
for
the
MMP-9
ribozyme.
Loading
in
each
lane
was
shown
to
be
equivalent
by
reprobing
the
blot
for
rPL32
mRNA,
a
gene
coding
for
a
ribosomal
protein.38'39
Thus,
introduction
of
the
ribozyme
directed
against
rat
MMP-9
resulted
in
de-
ceased
levels
of
MMP-9
mRNA.
The
decreased
levels
of
MMP-9
mRNA
in
cells
trans-
fected
with
the
ribozyme
against
MMP-9
were
reflected
in
decreased
levels
of
MMP-9
gelatinase
released
into
the
medium.
(Figure
3).
Gelatinase
activity
at
105
kd
as
de-
tected
by
substrate
gel
electrophoresis
indicated
that
the
parental
148.1,LMD
cells
and
the
control
transfectants
secreted
equivalent
amounts
of
105-kd
gelatinase
activ-
ity,
but
conditioned
medium
from
the
two
ribozyme
trans-
fected
clones
had
5-
to
10-fold
less.
After
stimulation
with
either
TGF-,B
or
TPA,
the
parental
and
control
cells
re-
leased
greater
amounts
of
MMP-9
activity
into
the
me-
dium.
The
ribozyme
transfected
cells
also
released
greater
amounts
of
gelatinase
activity
after
stimulation,
but
the
absolute
level
was
still
considerably
reduced
in
comparison
with
the
controls.
No
lower
molecular
mass
bands
indicative
of
activation
were
seen
in
any
case.
To
determine
the
effect
of
lowered
MMP-9
expression
on
the
behavior
of
these
prostatic
carcinoma
cells,
we

594
Sehgal
et
al
AJP
February
1998,
Vol.
152,
No.
2
UMMp-9
ribozyme
A
Ib
2
q
Control
ribozyme
r
Ribozyme
Probe
rpL
32
MMP-9
Control
B
nbozyme
nbozyme
1.51
1.52
1.74
1.22
O
t
.
L
.
.
L-
.
<-
MMP-9
L
F
F
EL
<
u:(1:
UL
c<
a.
uLc-
-S
Parent
1.51
1.22
(Ribozyme)
(Control)
Ca
(DO<
OI-H
lLCC
O
a.
-
F- F-
ec
-
F- F-
Parent
1.52
1.74
(Ribozyme)
(Control)
Figure
3.
Ninety-two-kd
gelatinase
activity
is
reduced
in
conditioned
me-
dium
from
ribozyme-transfected
cells.
Conditioned
medium
was
collected
from
cells
treated
with
the
indicated
agent
and
subjected
to
gelatin
substrate
gel
electrophoresis.
r
p
L
3
2
Figure
2.
The
MMP-9
ribozyme
results
in
decreased
MMP-9
mRNA
expres-
sion.
A:
Expression
of
the
ribozymes
in
transfected
cells.
Poly(A)
plus
RNA
was
isolated
from
the
parental
cells
148-1,
from
cells
transfected
with
the
control
ribozyme
1.22
and
1.74,
and
from
the
MMP-9
ribozyme
1.51
and
1.52.
The
upper
panel
shows
the
autoradiograph
after
hybridization
with
a
probe
derived
from
the
ribozyme
expression
vector,
whereas
the
lower
panel
shows
a
loading
control
with
the
same
blot
reprobed
using
rPL32.
B:
Ex-
pression
of
MMP-9
mRNA
in
the
ribozyme-transfected
cells.
Total
RNA
(10
,xg)
from
each
of
the
indicated
cells
treated
with
TGF-(3
or
with
TPA
were
subjected
to
Northern
blotting
with
an
MMP-9
probe.
The
upper
panel
shows
this
autoradiograph,
and
the
lower
panel
shows
the
autoradiograph
after
reprobing
with
a
loading
control
rPL32.
tested
their
metastatic
potential
using
the
lung
coloniza-
tion
assay
(Table
1).
After
injection
of
5
x
105
cells
into
the
tail
veins
of
female
nude
mice,
both
the
parental
cells
and
the
controls
gave
rise
to
lung
colonies
(22
to
31
per
mouse),
but
injection
of
the
same
number
of
MMP-9
ribozyme
transfected
cells
gave
rise
to
at
most
1
to
3
per
mouse.
Although
the
rasH
plus
myc-transformed
prostate
cells
derived
from
the
murine
prostate
reconstitution
sys-
tem
have
been
reported
to
be
androgen
independent,
we
also
wished
to
determine
whether
the
cells
would
re-
spond
differently
after
injection
in
a
male
mouse.40
The
experimental
metastasis
assay
was
performed
using
male
nude
mice.
In
this
case
the
controls
and
parental
cells
again
readily
gave
rise
to
lung
colonization,
from
28
to
35
nodules/mouse,
whereas
only
3
to
4
nodules/mouse
were
seen
after
injection
of
the
clones
with
down-regu-
lated
MMP-9.
Thus,
metastasis
was
inhibited
by
down-
regulation
of
MMP-9.
Tumor
growth
rate
was
evaluated
after
subcutaneous
injection
in
male
nude
mice
with
no
significant
differences
seen
regardless
of
their
MMP-9
expression
(Figure
4).
Discussion
The
metastatic
potential
of
the
prostate
cells
with
re-
duced
MMP-9
was
drastically
reduced
compared
with
either
control
transfected
clones
or
the
parental,
meta-
static
cells.
In
contrast,
tumor
growth
was
unaffected.
The
finding
that
MMP-9
contributes
to
metastasis
in
a
murine
prostate
model
system
leads
to
the
question
of
whether
MMP-9
might
be
involved
in
tumor
progression
in
human
Table
1.
Effect
of
MMP-9
Ribozyme
on
Experimental
Metastasis
Male
(lung
nodules/
Female
(lung
nodules/
Cell
type
Cell
lines
mouse
±
SD)
mouse
±
SD)
Parental
148-1,LMD
29
±
8.5
26.2
±
5.3
Control
ribozyme
AAS
1.22
37
±
11
22.2
±
6.7
AAS
1.74
38
±
8
31.8
±
9.9
MMP-9
Ribozyme
AR
1.51
3.25
±
2.5
0.63
±
0.3
AR
1.52
3.0
±
2.2
1.5
±
0.5
Cells
(5
x
104)
from
the
parental
population,
cells
from
two
MMP-9
ribozyme
transfected
clones
AR1.51
and
AR1.52,
and
from
two
control
transfectants
AAS
1.22
and
AAS
1.74
were
each
injected
intravenously
into
male
or
female
nude
mice.
After
4
weeks,
the
animals
were
sacrificed
and
the
number
of
lung
nodules
scored.
The
results
shown
are
from
one
experiment
with
five
mice
in
each
group.
Similar
results
were
obtained
in
two
repeat
experiments.
The
range
of
nodule
number
in
all
experiments
for
the
cells
in
the
parental
group
was
from
12
to
55,
for
the
control
ribozyme
cells
from
22
to
82,
and
for
the
MMP-9
ribozyme
cells
from
0
to
3.
The
difference
between
the
results
from
the
control
ribozyme
and
the
MMP-9
ribozyme
groups
is
significant
to
P
<
0.001
by
the
Mann-Whitney
test.

MMP-9
in
Prostate
Cancer
595
AJP
February
1998,
Vol.
152,
No.
2
I>---
1.74
Control
Ribozyme
0.8-
---0O----
1.22
---
1.51
MMP-9
-YD-w
---
1.52
Ribozyme
T
.2.
0.6
~~~~
Parental
E
cE
4
0
Tj
0
E
0.2-
2.5
5
7.5
10
12.5
15
Time
(days)
Figure
4.
Tumor
growth
rate
is
not
affected
by
MMP-9
expression.
The
diameter
of
the
tumors
formed
after
injection
of
the
indicated
cells
was
measured.
The
standard
deviation
is
shown
with
error
bars.
When
the
error
bars
cannot
be
seen,
they
were
smaller
than
the
diameter
of
the
data
point.
prostate
cancer.
Wood
et
al41
found
low
levels
of
MMP-9
mRNA
in
normal
prostate
tissue,
tissues
from
patients
with
benign
prostatic
hypertrophy,
or
carcinoma
with
a
low
Gleason
score
(5
or
less).
In
contrast,
MMP-9
mRNA
levels
were
highly
expressed
in
specimens
from
carcino-
mas
with
high
Gleason
scores,8-10
and
there
was
a
sig-
nificant
correlation
with
high
MMP-9
expression
and
local
invasion
as
well
as
survival.
Elevated
MMP-9
levels
were
found
in
cell
cultures
from
prostate
carcinomas
with
high
Gleason
scores,42
and
gelatinase
profiles
from
tissue
samples
revealed
a
correlation
with
MMP-9
gelatinase
activity
and
metastasis.43
These
data
are
consistent
with
MMP-9
filling
a
role
in
human
prostate
cancer
similar
to
its
role
in
the
mouse
reconstitution
model
of
prostate
cancer.
Other
MMPs
have
also
been
implicated
as
playing
a
role
in
the
behavior
of
prostate
carcinoma.
MMP-7
(ma-
trilysin
or
PUMP-1)
has
been
found
to
be
overexpressed
in
the
epithelial
cells
of
a
high
frequency
of
human
pros-
tate
carcinoma
specimens
and
prostatic
carcinoma
cell
lines.544
Transfection
of
an
expression
vector
for
matri-
lysin
into
the
prostatic
carcinoma
line
DU
145
resulted
in
increased
invasion
into
the
diaphragm
by
these
cells,
and
this
correlated
with
invasion
in
the
prostate
itself.
MMP-2
(gelatinase
A
or
72-kd
type
IV
collagenase)
is
also
overexpressed
in
prostatic
carcinoma
cells
indicat-
ing
that
it
too
may
play
an
important
role
in
the
biological
behavior
of
this
tumor.
Whether
TGF-,3
acts
in
vivo
to
stimulate
MMP-9
expres-
sion
and
thus
lead
to
metastatic
behavior
is
an
unre-
solved
issue
at
this
point.
TGF-,3
is
expressed
at
higher
levels
in
prostatic
carcinoma
specimens
from
patients
with
more
aggressive
disease.4548
It
has
also
been
re-
ported
that
latent
TGF-,j
binding
protein
is
absent
from
prostatic
carcinoma
specimens
but
present
in
normal
tissues
and
those
from
patients
with
benign
diseases.49
It
has
also
been
suggested
that
response
of
TGF-,3
recep-
tor
1
may
be
altered
in
some
prostatic
carcinomas
po-
tentially
leading
to
differential
responses
to
TGF-f3;
al-
though
this
does
not
appear
to
be
the
case
in
the
mouse
prostate
reconstitution
system
as
both
receptors
were
present
in
cells
derived
from
these
tumors,
and
TGF-f
led
to
equivalent
plasminogen
activator
inhibitor
1
stimulation
in
metastatic
and
nonmetastatic
cells.3,50
The
effects
of
TGF-p,
however,
must
be
pleiotrophic
as
its
effects
on
cell
growth
are
likely
to
be
independent
of
its
effects
on
MMP-9
production.
These
results
complement
our
previous
results
in
a
murine
sarcoma
model
system
in
which
down-regulation
of
MMP-9
also
led
to
a
decrease
in
metastatic
potential
without
a
decrease
in
tumorigenicity
or
tumor
growth
rate.
Whereas
it
has
often
been
assumed
that
MMPs
were
involved
in
metastasis,
these
studies
now
begin
to
pro-
vide
some
evidence
that
MMP-9
in
fact
does
play
a
role
in
metastasis.
As
MMP-9
is
overexpressed
in
many
hu-
man
tumor
types,
these
results
raise
the
possibility
that
MMP-9
expression
may
be
linked
to
tumor
progression
and
to
the
development
of
metastatic
potential
in
a
variety
of
human
cancers,
including
prostate
cancer,
and
that
MMP-9
expression could
be
a
potential
target
for
the
modulation
of
metastatic
potential.
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T,
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1998,
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