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* Departments of Medicine and Oncology, McGill University and Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1;
Department of Pathology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;
Department of Clinical Biochemistry, Haddassah Medical School, Jerusalem, Israel IL-91120;
§ Institute for Cancer Research and Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Ft. Worth, Texas 76107, USA;
** Department of Biology; Ångstrom Pharmaceuticals Inc., San Diego, California 92121, USA; and
Northwestern University School of Medicine, Chicago, Illinois 60611, USA
2Correspondence: McGill University Health Center, Room H4.67, 687 Pine Ave. West, Montreal, Quebec, Canada H3A 1A1. E-mail: srabbani{at}med.mcgill.ca; Å6 correspondence should be addressed to: T.R.J., mazar{at}angstrominc.com
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ABSTRACT |
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 TOP ABSTRACT UROKINASE, BREAST CANCER,...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • breast cancer • endothelial cell • tumor necrosis
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UROKINASE, BREAST CANCER, ANGIOGENESIS, AND APOPTOSIS |
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TOPABSTRACTUROKINASE, BREAST CANCER,...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. Expression of the components
of the uPA system and its specific cell-surface receptor (uPAR) often
increases with disease progression and is correlated with poor
prognosis and outcome in patients (5
, 6)
. The expression
of uPA and uPAR is not restricted to tumor cells alone since other
tumor-associated cells, such as angiogenic endothelial cells,
macrophages, and fibroblasts have been demonstrated to express one or
both components of this system (7
8
9)
. Moreover, the
pattern of expression and the cells responsible for this expression may
differ depending on the type and stage. Expression of uPA and uPAR is
associated with tumor progression and is often localized to the
leading, invasive edge of a tumor (10
, 11)
.
The uPA system has pleiotropic functions in tumor progression whereby
several pathways may be temporally activated when uPA binds to uPAR.
Various intracellular signaling pathways are initiated when uPA binds
to uPAR including the up-regulation of oncogene expression, stimulation
of cell adhesion, regulation of chemotaxis, and activation of the MAP
kinase pathway (12
13
14
15
16)
. However, the mechanism of
signaling via uPAR, a glycolipid-anchored receptor that lacks a
transmembrane signaling domain, and the identity of the adapter
molecule(s) hypothesized to couple ligand binding to intracellular
signaling remain elusive. Receptor binding also results in the
activation of scuPA, the single-chain zymogen form of uPA, and
initiates an extracellular proteolytic cascade that leads to the
downstream activation of plasminogen and matrix metalloproteases
(17
, 18)
. These enzymes remodel extracellular matrix (ECM)
and the basal lamina associated with endothelial cells, and also
release or activate various growth factors sequestered within the ECM
such as vascular endothelial growth factor (VEGF), fibroblast growth
factor 2 (FGF-2), and transforming growth factor ß
(19
20
21)
. The net result of this proteolytic flux combined
with uPA-dependent intracellular signaling is acceleration of tumor
cell invasion and tumor-associated angiogenesis.
The primary interaction of uPA with uPAR is mediated through the growth factor domain amino acids (aa) 1–48 of uPA. However, we have identified a second site in uPA that interacts with uPAR. This region, termed the connecting peptide, is comprised of aa 136–143. Here we present data that a small, capped peptide derived from this region, Ac-KPSSPPEE-Am (Å6), inhibits the interaction of uPA with uPAR in a noncompetitive manner. Administration of this peptide to animals bearing experimental breast cancer tumors results in inhibition of tumor growth and metastasis in the absence of direct cytotoxic or anti-proliferative effects.
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MATERIALS AND METHODS |
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TOPABSTRACTUROKINASE, BREAST CANCER,...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determination of Å6 dose for in vivo studies
Single administrations of Å6 up to 1500 mg/kg did not
demonstrate any evidence of toxicity. A liquid chromatography/mass
spectroscopy assay was developed to detect Å6 in plasma.
Pharmacokinetic experiments demonstrated a short plasma half-life for
Å6 (0.2 h) in mice. We initially attempted to achieve a plasma level
at steady state (CSS) that was at least
equivalent to the in vitro IC50 for
tumor cell invasion (5–10 µM). This objective was accomplished by
delivering Å6 via continuous infusion using osmotic mini-pumps at a
dose of 75 mg · kg-1 · day-1. We then
compared administration of this dose by continuous infusion or by twice
daily (bid) injections. Both protocols yielded identical
anti-tumor results, indicating that the pharmacokinetics did not
correlate with the anti-tumor activity of Å6 and that a steady-state
plasma level of Å6 was not required for anti-tumor activity. For
simplicity, the IP protocol was used for all subsequent studies.
Vehicle (phosphate-buffered saline, or PBS) was used as the control for in vivo studies since it is relevant to the therapeutic setting. Although scrambled peptides are useful for establishing specificity in vitro, they are completely different chemical entities and most likely have different behavior in vivo. Thus, they were not considered as controls for the animal studies.
Cells and cell culture
The rat mammary adenocarcinoma cell line Mat B-III was obtained
from the American Type Culture Collection (ATCC, Rockville, Md.). Cells
were maintained in culture in vitro in McCoy’s 5A modified
medium (Gibco BRL, Burlington, ON, Canada) supplemented with 10% fetal
bovine serum (FBS), 25 mM 4-(2-hydroxyethy)-1-piperazineethanesulfonic
acid, 26 mM sodium bicarbonate, 100 units/ml of penicillin-streptomycin
sulfate (Gibco BRL), and 0.2% gentamicin (Sigma, St. Louis, Mo.).
Human breast adenocarcinoma cell line MDA-MB-231 was obtained from ATCC and maintained in L-15 medium (Gibco BRL) supplemented with 10% FBS, 100 units/ml of penicillin-streptomycin sulfate, and 0.2% gentamicin. MDA-MB-231-GFP (green fluorescent protein) cells were generated by transfecting the expression vector that contained the codon-optimized human GFP-S65T gene (Clontech Laboratories, Inc., Palo Alto, Calif.) using Lipofectin reagent (Gibco BRL). Cells with stably integrated plasmids were selected for neomycin resistant gene with geneticin (G418) (Gibco BRL).
Surface plasmon resonance
Binding kinetics of suPAR in the presence and absence of Å6 was
measured using a BIA 3000 optical Biosensor (Biacore, AB, Sweden)
(22)
. This method detects binding interactions in real
time by measuring changes in the refractive index (RI) at a biospecific
surface, and enables association and dissociation rate constants to be
calculated. For these studies, recombinant scuPA (a kind gift of Dr.
Jack Henkin, Abbott Laboratories, Abbott Park, Ill.) was coupled to a
B1-research grade sensor chip flow cells (Biacore, AB, Sweden) via a
standard amine coupling procedure (23)
using
N-hydroxysuccinimide/N-ethyl-N-[3-(dimethylamino) propyl]
carbodiimide hydrochloride (Pierce, Rockford, Ill.) at a level of 1000
RU each. Sensor surfaces were coated with ligands (10 µg/ml) in 10 mM
NaOAc buffer, pH 5.0. After immobilization, unreacted groups were
blocked with 1 M ethanolamine, pH 8.5. Binding buffer was PBS, pH 7.4,
0.005% TWEEN-20. Binding of suPAR (± Å6) was measured at 25°C at a
flow rate of 100 µl/min for 1 min, with 2 min of dissociation
examined. The bulk shift due to changes in RI was measured using the
suPAR surface and was subtracted from the binding signal at each
condition to correct for nonspecific signals. Surfaces were regenerated
with 2 x 30 s pulses of 1 M NaCl, pH 3.3, followed by an
injection of binding buffer for 1 min to remove this high-salt
solution. All injections were performed in a random fashion using the
RANDOM command in the automated method. Binding of suPAR was performed
at 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, and 1.24 nM in the absence or
presence of 60 µM, 20 µM, 6.6 µM, 2.2 µM, 0.74 µM, or 0.25
µM Å6. Data were fit using a 1:1 Langmuir reaction mechanism using
BIA evaluation 3.0 software (Biacore, AB, Sweden). Dissociation and
association rates were calculated separately to examine the effect of
Å6 on the affinity of suPAR to scuPA. Clot lysis assay was performed
as described previously (24)
.
Boyden chamber Matrigel invasion assay
The effect of Å6 on-treated Mat B-III and MDA-MB-231-GFP cells
was determined by two compartment Boyden chambers (Transwell, Costar,
Cambridge, Mass.) and basement membrane Matrigel invasion assay as
described previously (25)
. The 8 µm pore polycarbonate
filters were coated with basement membrane Matrigel (50 µg/filter).
Matrigel was then reconstructed by adding 0.1 ml serum-free culture
medium to the upper chamber and incubated for 90 min. After removal of
medium, cells (5x104) in 0.1 ml of medium with
or without Å6 was added to the upper chamber and placed in a lower
chamber prefilled with 0.8 ml of serum-free medium supplemented with 25
µg/ml fibronectin (Sigma, Oakville, ON, Canada) and incubated at
37°C for 24 h. At the end of incubation, medium was removed and
filters were fixed in 2% paraformaldehyde, 0.5% glutaraldehyde
(Sigma) in 0.1 M phosphated buffer, pH 7.4 at room temperature (RT) for
30 min. After washing with PBS, all filters were stained with 1.5%
toluidine blue and filters were mounted onto glass slides. Cells were
examined under a light microscope. Ten fields under 400x magnification
were randomly selected and the mean cell number was calculated.
Endothelial cell migration assay
Transwell (Costar, 8.0 µm pore size) were coated with type I
collagen (50 µg/ml) by adding 200 µl of the collagen solution per
transwell, then incubating overnight at 37°C. The transwells were
assembled in a 24-well plate and bFGF (1 ng/ml) was added to the bottom
chamber in a total volume of 0.8 ml M199 containing 2% FBS. Human
dermal microvascular endothelial cells were detached from monolayer
culture using Verseen, centrifuged, and reconstituted in M199
containing 2% FBS (1x106 cells/ml). 0.2
milliliters of this cell suspension was added to the upper chamber of
each Transwell. Inhibitors to be tested were added to both the upper
and lower chambers, and the migration was allowed to proceed for 5 h under 5% CO2 in a humidified atmosphere at
37°C. The transwells were then removed from the plate and the upper
chamber wiped clean with a cotton swab. Giemsa stain was used to fix
and stain the cells, and the number of cells that had migrated to the
bottom aspect of the membrane was counted. Data are presented as the
average number of migrated cells per 10 fields.
Animal protocols
Inbred female Fischer 344 rats weighing 200–220 g were obtained
from Charles River Inc. (St. Constant, QC, Canada). Before inoculation,
Mat B-III tumor cells grown in serum-containing medium were washed with
Hank’s balanced buffer and trypsinized for 5 min. Cells were then
collected in Hank’s balanced buffer and centrifuged at 1500 rpm for 5
min. Cell pellets (1x106 cells) were resuspended
in 0.2 ml saline and injected using 1 ml insulin syringes into the
mammary fat pad of rats anesthetized with ethanol/Somnotal (MTC
Pharmaceuticals, Cambridge, ON, Canada). Control and experimental
animals were injected intraperitoneally (i.p.) with PBS and Å6 (75
mg·kg-1·day-1)
respectively twice a day for 16 days. All animals were numbered, kept
separate, and examined for the development of tumors daily for up to 17
days. The tumor mass of control and experimental animals was measured
in two dimensions by calipers and the tumor volume was calculated.
Control and experimental animals were killed at the end of this study
(day 17) and examined and scored for the development of macroscopic
tumor metastases in various tissues. Primary tumor tissues were also
removed from control and experimental animals for histological
examination.
For xenograft studies, 4- to 6-wk-old BALB/c (nu/nu) female mice were obtained from Charles River Inc. Prior to inoculation, MDA-MB-231-GFP cells grown in serum containing culture medium were washed with Hank’s balanced buffer and centrifuged at 1500 rpm for 5 min. Cell pellets (5x105 cells/mice) were resuspended in 100 µl of Matrigel (Becton Dickinson Labware, Mississauga, ON, Canada) and saline mixture (20% Matrigel) and injected into the mammary fat pads of the mice. All animals were numbered and kept separately in a temperature-controlled room on a 12 h/12 h light/dark schedule with food and water ad libitum. Tumors were allowed to grow to the size of 15–25 mm3 prior to drug administration. At this time, animals were randomized into two groups. Å6 (75 mg·kg-1·day-1) and sterile PBS were injected i.p. twice a day into the two groups of mice, respectively. The tumor mass was measured in two dimensions with calipers twice a week. At the end of xenograft study, the mice were killed and their lungs, liver, spleen, and other organs were removed. These fresh tissues from control and experimental mice were sliced at ~1 mm thickness and observed directly under a fluorescence microscope. The number of tumor cells from 10 random sites was counted per field of examination and photographed.
Histology and immunohistochemistry
Primary tumors were fixed in 4% paraformaldehyde overnight,
dehydrated, and embedded in paraffin (Fisher Scientific, Montreal, QC,
Canada) the next day. Tumor sections (4 µm) were deparaffinized, then
rehydrated and stained with hematoxylin/eosin (H&E). For TUNEL assay,
paraffin-embedded tissue sections were soaked in Toluene to
deparaffinize, then rehydrated in graded alcohol series (100% to
70%). For enzyme predigestion of formalin-fixed tissue, the sections
were incubated for 30 min at RT in 15 µg/ml of proteinase K/10 mM
Tris/HCl. The terminal deoxynucleotidyl transferase (Boehringer
Mannheim, QC, Canada) diluted in TUNEL reaction mixture, was added to
the slides and incubated at 37°C for 60 min. After rinsing with PBS,
the sections were coated with 50 µl of anti-fluorescein antibody
conjugated with converter-POD (horseradish peroxidase) at 37°C for 30
min. For factor VIII staining enzyme predigestion of formalin-fixed
tumors was done by incubation at 37°C in 0.1 g Pronase type
14/100 ml PBS, followed by washing in 10 mM PBS (pH 7.6). Anti-human
endothelial cell antibody against factor VIII-related antigen (Von
Willebrand factor) (DAKO Diagnostica Inc., Mississauga, Ontario,
Canada) was used as primary antibody diluted in serum/PBS (1:600).
Tumor sections were incubated for 60 min at RT, followed by further
incubation for 30 min with biotinylated anti-rabbit link antibody
(Zymed Laboratories Inc., San Francisco, Calif.). Sections were
rinsed with PBS, followed by coating with streptavidin conjugated to
horseradish peroxidase for 10 min. The substrate DAB was then incubated
with TUNEL and factor VIII-related antigen sections for 10 min at RT
(Sigma, Ontario, Canada). Finally, the sections were counterstained
with hematoxylin and mounted. All histological examinations were
carried out by light and fluorescence microscopy using a Nikon
microscope equipped with a Xenon lamp power supply and a GFP filter set
(Chromotechnology Corp., Brattleboro, Vt.). For quantitation of
TUNEL-positive cells, three sections from each tumor were analyzed
using NIH image version 1.61 and expressed as integrated density per
field of examination. In control and Å6-treated tumors, microvessels
were counted and expressed as angiogenesis density representing the
mean of at least three areas with high vascularization in three
different sections from each tumor. All slides were interpreted by two
independent investigators (26)
.
Statistical analysis
Results are expressed as the mean ± SE of at
least triplicate determinations and statistical comparisons are based
on Student’s t test or analysis of variance. A probability
value of <0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTUROKINASE, BREAST CANCER,...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Similar results were observed at 33.3 nM and 11.1 nM
suPAR (data not shown). The specificity of this inhibition was
demonstrated by the lack of effect of Å29, an analog of Å6 in which
the first serine is replaced by a glutamic acid (data not shown). This
amino acid substitution was chosen because it mimics a phosphorylated
serine at this position. Franco et al. (27)
have described
that high molecular weight uPA (HMW uPA) can be phosphorylated at this
serine and that phosphorylated uPA is unable to mediate monocyte
chemotaxis or adhesion, in contrast to unmodified uPA. Association and
dissociation rate constants were calculated for suPAR binding to scuPA
in the absence and presence of Å6 at the three highest concentrations
of suPAR. The kd and the
ka were calculated separately for each
of 21 conditions and the data were fitted to a 1:1 Langmuir model.
Langmuir isotherms in the absence and presence of Å6 were not
parallel, suggesting that Å6 did not inhibit the suPAR–scuPA
interaction in a competitive manner (data not shown). The
Kd of suPAR was 1 nM and 1.7 nM in the absence and
presence of Å6, respectively, whereas the Rmax
decreased in a dose-dependent manner (Fig. 1B
, 100 nM
suPAR). The maximal inhibition observed was 50%, indicating that a new
steady state was established in the presence of Å6. Increasing
concentrations of Å6 did not significantly alter the
Kd of suPAR for scuPA, and therefore it was not
necessary to carry out a Schild regression because it was evident that
the inhibition was not competitive. Overall, the kinetic and
equilibrium binding constants were not altered by Å6
[kd =
(1.0x10-3 ± 1.1x10-4)
s-1, ka =
(7.5x105 ± 7.0x104)
M-1·s-1, and the
Kd = (1.3x10-9 ±
2.9x10-10) M]. In contrast, 10 nM soluble
scuPA was able to completely inhibit the binding of equimolar
concentrations of suPAR. Soluble scuPA (10 nM) decreased the apparent
affinity of suPAR to immobilized scuPA (100 nM suPAR,
Kd = 2.6x10-9 M, 33.3 nM
suPAR, Kd = 2.6x10-7 M), as
would be expected for a competitive inhibitor. These data are
consistent with Å6 inhibiting the suPAR–scuPA interaction in a
noncompetitive, allosteric manner.
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Additional evidence for the allosteric inhibition of the scuPA–suPAR
interaction by Å6 was derived from the finding that Å6 inhibits
plasminogen activation by the scuPA-suPAR complex in a clot lysis assay
(28)
. The plasminogen-activating activity of this complex
has been hypothesized to result from a conformational change in the
complex that leads to the formation of an active site in the absence of
conversion to two-chain uPA (tcuPA) (28)
. Å6 did not
inhibit clot lysis initiated by tcuPA (data not shown). However, Å6
almost completely abrogated clot lysis mediated by the scuPA-suPAR
complex whereas the control peptide Å29 had no such effect (Fig. 1C
).
Å6 inhibits breast cancer cell invasion and endothelial cell
migration in vitro
Tumor cell invasion and endothelial cell migration during
angiogenesis are key events that contribute to tumor progression. The
uPA–uPAR system has been demonstrated to play a major role in both
processes. Since Å6 inhibited the interaction of suPAR with scuPA, we
assessed its ability to inhibit cell invasion and migration in
vitro. Peptides of different length based on the connecting
peptide region of uPA (aa 136–158) were prepared and tested for their
ability to inhibit MDA-MB-231 tumor cell invasion in vitro.
Of all the peptides tested, only Å6 was able to inhibit tumor cell
invasion in this assay (Fig. 2A
). Uncapped versions of Å6 also had no activity, indicating
that Å6 represented the minimal active epitope of the connecting
peptide, at least from the standpoint of invasion (Fig. 2A
).
The dose dependence of Å6 inhibition of rat and human breast cancer
cell invasion was evaluated in a Boyden chamber invasion assay. Å6
decreased the number of rat and human breast cancer cells invading
through Matrigel with an IC50 between 5–25 µM
for both cell lines tested (Fig. 2B, C
). Invasion was not
inhibited further at Å6 concentrations greater than 50 µM. In
contrast to molecules that inhibit the binding of the GFD (aa 1–48) of
uPA to uPAR (29)
, the ability of Å6 to inhibit invasion
did not appear to be species specific. Finally, Å6 also inhibited the
migration of human dermal microvascular endothelial cells on type I
collagen (Fig. 2D
). The IC50 for
endothelial cell migration (25–50 µM) was slightly higher than that
observed in the tumor cell invasion assay.
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Å6 blocks rat breast cancer growth and metastasis in
vivo
Since the uPA–uPAR system contributes to the invasion and
motility of multiple cell types associated with tumor progression
(e.g., tumor cells, endothelial cells), we hypothesized that the
inhibition of the uPA–uPAR interaction using Å6 would have
significant anti-tumor effects in vivo. We expected that a
capped peptide would be less susceptible to exoprotease degradation in
the plasma (although this was not specifically tested) and would
therefore have a half-life sufficient to provide efficacy in
vivo. Rat Mat B-III breast cancer cells
(1x106 cells) were inoculated into the mammary
fat pads of female Fischer rats. Å6 treatment (75
mg·kg-1·day-1) was
initiated on the same day as the inoculation and continued for 16 days.
Tumor volumes were determined daily and compared to the control group,
which received vehicle alone. Infusion of Å6 resulted in a marked
decrease (55%) in tumor volume throughout the course of this study
(Fig. 3A
). Control and Å6-treated animals were killed on day 17 and
evaluated for the presence of macroscopic tumor metastases. Control
animals, receiving vehicle alone, developed macroscopic tumor
metastases to lungs, retroperitoneal and axillary lymph nodes. In
contrast, all Å6-treated animals exhibited significantly fewer or no
macroscopic tumor metastases at these sites (Fig. 3B
).
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Å6 blocks human breast cancer growth and metastasis in
vivo
We then examined the anti-tumor effects of Å6 in a xenograft
model of human breast cancer. To facilitate the detection of metastatic
tumor cells, we first transfected human breast cancer cells
(MDA-MB-231) with the cDNA for green fluorescent protein
(MDA-MB-231-GFP). MDA-MB-231 and MDA-MB-231-GFP cells exhibited no
significant difference in their invasive capacity in a Matrigel
invasion assay (data not shown). For in vivo studies,
MDA-MB-231-GFP cells (5x105) were coinoculated
into the mammary fat pads of female BALB/c (nu/nu) mice with
Matrigel. Tumors were allowed to grow until palpable (15–25
mm3, ~4 wk after inoculation), at which time
Å6 administration was initiated. Animals received Å6 (75
mg·kg-1·day-1) or
vehicle alone for 6 wk and tumor volumes were determined at weekly
intervals. Administration was discontinued after 6 wk and the animals
were killed and evaluated at the end of week 7. Å6 administration
resulted in a significant (-90%) decrease in the primary tumor volume
(Fig. 3C
), which was clearly evident upon visual inspection
of the dead animals (Fig. 3D
). Necropsy examination revealed
that control animals receiving vehicle alone routinely developed a
large number of macroscopic tumor metastases in the lungs and axillary
lymph nodes. In contrast, Å6 treatment resulted in significantly fewer
and smaller metastases to lymph nodes and a trend to decreased
macroscopic metastases to lungs (Fig. 3E
). We also evaluated
the number of microscopic tumor foci present in other organs (liver,
lungs, and spleen). Microscopic dissemination was observed only in
these organs, and the number of metastatic tumor cells was
significantly lower in the liver and lungs of Å6-treated animals when
compared to vehicle controls (Fig. 3F
). No significant
difference in the number of disseminated tumor cells was observed in
the spleens of control and Å6-treated group of animals.
Å6 treatment leads to extensive tumor necrosis
Necrotic cores are often observed in rapidly growing experimental
tumors, and we observed this in both the syngeneic (Mat B-III) and the
xenograft (MDA-MB-231-GFP) models. Although tumor core necrosis
(~20% of the total tumor volume) might be expected to occur as a
result of hypoxia, this is not the case at the periphery of the tumors.
The periphery of primary Mat B-III control tumors appeared
predominantly viable when assessed by H&E staining (Fig. 4a
). In contrast, H&E staining of sections from Å6-treated
tumors revealed large areas of hemorrhagic necrosis (in this context,
necrosis is used to describe tumor cell death regardless of the
mechanism or pathway involved in that process), which comprised greater
than 75% of the peripheral tumor area (Fig. 4b
). TUNEL
staining of tumor sections revealed very few TUNEL-positive cells in
sections obtained from control Mat B-III tumors (Fig. 4c
).
However, sections obtained from Å6-treated tumors revealed extensive
positive staining by TUNEL (Fig. 4d
). TUNEL-positive foci
were significantly more numerous in Å6-treated animals and were
observed in both necrotic and non-necrotic regions, suggesting that one
mechanism that could be responsible for cell death was apoptosis (Fig. 4
, bottom). Å6 treatment of tumor cells did not inhibit proliferation
or directly lead to apoptosis in vitro (data not shown).
Similar results were also observed in sections obtained from
MDA-MB-231-GFP xenograft tumors (data not shown).
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Å6 treatment results in decreased tumor vessel hot spots
Based on the ability of Å6 to inhibit endothelial cell
migration in vitro, we decided to evaluate whether Å6 had
any effect on tumor vessel formation in vivo. We used
anti-factor VIII-related antigen to stain both rat and mouse blood
vessels in our respective models. Sections from Å6-treated tumors had
significantly less factor VIII-positive hot spots than sections from
control Mat B-III tumors (Fig. 5
). Similar results were observed in sections obtained from the
MDA-MB-231-GFP xenograft model (data not shown).
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|
DISCUSSION |
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TOPABSTRACTUROKINASE, BREAST CANCER,...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this report, we describe the activity of a peptide, Å6, derived
from the connecting peptide region of uPA that is capable of inhibiting
both of these cascades simultaneously through its inhibition of the
uPA–uPAR interaction. Since Å6 is derived from the connecting peptide
(aa 136–143) of uPA, it suggests that this region of uPA may represent
a novel epitope involved in the uPA–uPAR interaction. Franco et al.
(27)
recently demonstrated that Ser-138 (corresponding to
Ser-3 in the Å6 sequence) is phosphorylated in HMW uPA and that this
phosphorylation abrogates the ability of phosphorylated HMW uPA to
mediate monocyte chemotaxis. From the standpoint of drug development,
Å6 is especially intriguing since it apparently inhibits the uPA–uPAR
interaction in an allosteric manner, perhaps by altering or stabilizing
a particular conformation of one or both of these proteins. Å6 does
not inhibit the growth of any cell line tested thus far in
vitro, including tumor and endothelial cells. However, despite
this lack of direct cytotoxic or anti-proliferative activity, Å6
treatment leads to a striking suppression of tumor growth and
metastasis in several animal models using breast cancer cell lines.
Å6 inhibits the tumor growth of the MDA-MB-231 cells to a greater
extent than the Mat B-III cells in vivo. Mat B-III tumors
grow much faster than the MDA-MB-231 tumors in vivo and may
overcome the inhibitory effects of Å6. Our data suggest that Å6
maintains tumor dormancy, and a rapidly proliferating tumor such as the
Mat B-III might overcome this suppressive effect much more rapidly than
a more indolent tumor such as the MDA-MB-231. This seems to be an
emerging paradigm for anti-angiogenic agents when they are used as
monotherapy in preclinical models, as no single anti-angiogenic agent
(including angiostatin and endostatin) is able to reduce tumor burden
once tumors have reached 100 mm3
(30)
The MDA-MB-231 model may be more relevant to human
disease as most human solid tumors (including breast) are not highly
proliferative. We also present in vitro data on the ability
of Å6 to inhibit tumor cell invasion and human endothelial cell
migration, and the differences observed with Å6 treatment in
vivo could reflect species specificity. The high-affinity binding
of uPA to uPAR (via its growth factor domain) is species specific and
the affinities of the uPA for uPAR across species (for example, human
uPA binding to rat uPAR) differ by at least two orders of magnitude.
However, Å6 may represent a secondary, weaker affinity interaction
between uPA and uPAR that modulates conformation rather than direct
binding, and it is not known whether this interaction is species
specific. In addition, our data on the effects of Å6 on the uPA–uPAR
interaction have only been obtained using pure proteins and have not
been extended to cell systems. Therefore, the activity of Å6 against
tumor cells or endothelial cells could occur via some
non-uPAR-dependent mechanism as well.
Å6 is derived from the human uPA sequence, and the corresponding
sequences from rat and mouse are KPSSTVDQ and KPSSSVDQ. Since the KPSS
is conserved in all species, it is tempting to speculate that this may
represent the biologically active sequence of the peptide. We
demonstrate in Fig. 2A
that deletion of the amino-terminal
lysine abolished the anti-migratory effect of Å6, indicating that the
correct amino-terminal amino acid is important for the activity of Å6.
However, similar results were also observed when the carboxyl-terminal
amino acid was removed. The carboxyl-terminal amino acid is
semiconserved (Gln in rat and mouse and Glu in human) and perhaps this
is sufficient to maintain activity of the peptide. However, it is
difficult to draw conclusions regarding species specificity based on
the data presented in this report without knowing the contribution of
each amino acid to activity.
Å6 may inhibit metastasis in several interdependent ways. Inhibition of tumor growth may lead directly to decreased metastasis simply through a reduction in tumor burden. Smaller tumors may be less vascularized, affording less opportunity for hematogenous dissemination. Å6 may also directly suppress the outgrowth of disseminated tumors cells once they have seeded. Disseminated tumor foci also depend on angiogenesis to survive and proliferate. The anti-angiogenic effects of Å6 described below could suppress the ability of small tumor foci to recruit blood vessels necessary for survival. The inhibition of metastasis by Å6 could also result from direct anti-invasive effects on the tumor cells themselves. The ability of tumor cells to invade depends on multiple cellular activities such as mobility, adhesion, and cytoskeletal reorganization occurring in a temporally organized manner. Since Å6 is able to inhibit the invasion of tumor cells in vitro, it may interfere with one or more of these activities in vivo as well.
Our data also demonstrate that Å6 also has anti-angiogenic activity
resulting in substantially less factor VIII-positive hot spots in
sections obtained from Å6-treated animals. The effects of Å6 on
vascular contractility and signaling have also recently been
demonstrated by Haj-Yehia et al. (31)
, suggesting another
possible hypothesis to explain the activity of Å6. The inhibitory
effects of Å6 on vascular contractility may impede blood flow to the
tumor in addition to its effects on angiogenesis. Tumor neovessels,
which are prone to collapse and occlusion, may be especially
susceptible to an agent, such as Å6, that dysregulates vascular tone.
Aggressive, rapidly growing tumors contain large zones of hypoxic
cells. Low oxygen tension results in the up-regulation of both
vasoconstricting (e.g., endothelin-1) as well as angiogenic (VEGF,
PDGF) growth factor expression by endothelial cells (28)
.
Hypoxia has been implicated as a catalyst for the initiation of tumor
angiogenesis (32)
and may mediate both new vessel
formation (long term effect) as well as local vascular contractility
within a tumor. The fact that Å6 antagonizes the activity of
vasoconstricting agents such as phenylephrine and endothelin-1 suggests
that part of its anti-tumor effects may result from a suppression of
hypoxia-dependent pathways of angiogenesis in addition to the
inhibition of vessel formation. Further, Å6 does not seem to affect
nonstimulated vessels as no effects on vasoconstriction were observed
by Haj-Yehia et al. (31)
in the absence of
vasoconstricting stimuli. Experiments are currently under way to
examine the effects of Å6 on the expression of hypoxia-related genes
and the initiation of angiogenesis in tumors (angiogenic switch). This
hypothesis is consistent with the data observed in tumor models in
which Å6 treatment must be started before a tumor reaches a certain
critical size, after which time angiogenesis has already initiated and
Å6 treatment alone is no longer sufficient to inhibit tumor
progression.
The inhibition of vessel formation through either an anti-angiogenic or
an anti-vascular mechanism of action could also lead to increased tumor
cell death through apoptosis or related mechanisms. Clinical agents
(e.g., combretastatin) that disrupt tumor blood flow have demonstrated
potent anti-tumor effects leading to tumor cell death
(33)
. Despite extensive TUNEL staining in sections derived
from Å6-treated tumors, we cannot conclude that this represents
apoptosis since any process that leads to DNA fragmentation would also
result in TUNEL-positive staining. Nevertheless, the fact that tumor
cell death (histological examination of tissues obtained from
Å6-treated animals revealed no evidence of toxicity or nontumor
necrosis) occurs in the absence of any direct cytotoxic effects points
to modulation of the tumor microenvironment by Å6 as one likely aspect
of its mechanism of action.
We are currently in the process of identifying more potent analogs of Å6. These will be used to further elucidate the molecular basis of the anti-tumor effects described in this manuscript. The effect of an allosteric inhibitor such as Å6 cannot be competed away by ligand, making such a compound especially useful for inhibiting receptor–ligand interactions such as the binding of uPA to uPAR.
|
ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 23, 1999.
Revision received December 15, 1999.
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REFERENCES |
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TOPABSTRACTUROKINASE, BREAST CANCER,...MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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