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Cell Cycle. 2009 March 1; 8(5): 683–688.

MMP-2 functions as an early response protein in ovarian cancer

metastasis

Hilary A. Kenny and Ernst Lengyel*

Departments of Obstetrics and Gynecology; Section of Gynecologic Oncology; Center for
Integrative Science; University of Chicago; Chicago, Illinois USA

Abstract
The most common site (80%) of ovarian cancer metastasis is the omentum, a large (15 × 10 × 2 cm)
peritoneal fold covering the small bowel. Because of the absence of model systems that accurately
reproduce the microenvironment of the human omentum, the biological mechanism of early ovarian
cancer metastasis is poorly understood. Using a new organotypic 3D culture of the omentum, we
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show that when cancer cells adhere, matrix-metalloproteinase (MMP)-2 is upregulated and
proteolytically activated in these cells. The activated MMP-2 cleaves the matrix proteins fibronectin,
vitronectin and collagen I into smaller fragments. The cleaved extra-cellular matrix (ECM) fragments
then facilitate and accelerate cancer cell adhesion and invasion by binding to their cognate integrin
receptors. In vivo inhibition of MMP-2 before adhesion by using a siRNA or a blocking antibody
significantly reduced the number of metastasis and tumor weight in a xenograft mouse model. After
metastasis had been established, blocking MMP-2 produced less of an effect. Our data identify tumor-
derived proteolytically active MMP-2 as an early regulator of ovarian cancer metastasis.

Keywords
ovarian cancer; metastasis; adhesion; invasion; MMP-2

Introduction
Most ovarian cancers are of epithelial origin, arising from the single layer of cells that cover
the ovary or the fallopian tube.1,2 Once an ovarian epithelial cell undergoes transformation, it
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detaches from the underlying basement membrane and metastasizes throughout the peritoneal
cavity, carried by the flow of peritoneal fluid. Tumor cells then adhere, migrate, proliferate
and invade into the peritoneal surface, but rarely invade deeply into the ECM3 (Fig. 1). This
pattern of dissemination is responsible for the typical clinical presentation of patients with
ovarian cancer. Generally, these patients have metastatic tumor nodules within the abdominal
cavity, but rarely have distant metastases. The detachment/floating/attachment/invasion
sequence of ovarian cancer metastasis differentiates it from other much more common cancers,
such as colon and breast cancer that mainly metastasize hematogenously or lymphatically. This
noteworthy difference in the metastatic process makes ovarian cancer very unique in its clinical
and molecular characteristics, and is one of the main reasons that paradigms developed for
these much more common cancers (e.g., the adenoma-to-carcinoma-progression) often do not
apply to ovarian cancer. The primary microenvironment for ovarian cancer cells at the
metastatic site is the mesothelial cell. These cells cover the peritoneum, the small and large
bowel serosa and the omentum (Fig. 1A) forming a low-friction, non-adhesive surface and

*Correspondence to: Ernst Lengyel; University of Chicago; Department of Obstetrics and Gynecology; Section of Gynecologic
Oncology; MC 2050; 5841 South Maryland Avenue; Chicago, Illinois 60637 USA; [email protected].
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selective barrier.4 Mesothelial cells express ECM proteins at their surface (i.e., towards the
peritoneal cavity) including collagen types I, III, IV, fibronectin, laminin and vitronectin.5-7
Mesothelial cells lie on an ECM composed mainly of collagen type I, but also fibronectin,
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vitronectin and a continuous basement membrane, which includes laminin and collagen type
IV.8 Within the omental and peritoneal ECM and below the basement membrane are
fibroblasts, which are sometimes called submesothelial stromal cells, and below the ECM are
adipocytes (Fig. 1A). A key to understanding ovarian cancer metastasis is to elucidate the
interaction of ovarian cancer cells with stromal cells and the ECM at the secondary site.

Our laboratory recently developed an organotypic 3D model of the peritoneum and omentum
that mimics the human tissue and allows a detailed study of the early events of metastasis9,
10 and cell-cell communication. This model was designed after careful analysis of normal
human omentum and peritoneum and is constructed of primary human cells (mesothelial cells,
fibroblasts, adipocytes) derived from human tissue. After surgical removal of a piece of
omentum, the tissue is immediately digested and primary human fibroblasts or mesothelial
cells established. The fibroblasts are then mixed with type I collagen, the most common
peritoneal ECM, and a confluent layer of mesothelial cells is added on top (Fig. 2C). In order
to study cellular adhesion or invasion, primary cultures of ovarian cancer cells are
fluorescently-labeled and added to the 3D culture. The ovarian cancer and 3D model were then
co-cultured for 2–24 hours, and the molecular changes involved in adhesion and invasion were
investigated.12 Using this culture we found that human mesothelial cells play a protective role
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by actually inhibiting both the adhesion and invasion of ovarian cancer cells.9 This protective
role is also supported by a report showing that cancer cells have the ability to induce mesothelial
cell apoptosis. After the mesothelial cells detach, cancer cells are able to bind more efficiently
to the submesothelial basement membrane.11 Another important finding of this analysis was
that 4 hours after binding to the 3D model, the ovarian cancer cells upregulate and
proteolytically activate both MMP-2 and MMP-9.12 When we inhibited MMP-2 with siRNAs
or blocking antibodies, we found that cancer cell adhesion was reduced, while reduction of
MMP-9 had no effect on adhesion, indicating that MMP-2 was responsible for the increase
noted.12 These findings using the 3D culture were validated by other studies which found that
ovarian cancer cell attachment to full human omental tissue in vitro, and to mouse peritoneum
and omentum in vivo were also dependent on MMP-2.

We demonstrate here that the mechanism with which MMP-2 facilitates early adhesion and
invasion involves cleavage of multiple ECMs into smaller fragments that serve as better
attachment sites. In addition, we report that the inhibition of MMP-2, but not MMP-9,
significantly reduces ovarian cancer metastasis.

Results and Discussion

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Adhesion of ovarian cancer cells to different ECMs is facilitated by proteolytically active

MMP-2
MMP-2 is a protease that can cleave several ECM proteins; however the mechanisms by which
it facilitates cell adhesion are not clearly understood. The principal component of the
submesothelial ECM of both the peritoneum8,13 and omentum10 is composed of collagen type
I,14 vitronectin7 and fibronectin.5,6 In addition, mesothelial cells, themselves, produce collagen
type I, vitronectin and fibronectin protein.5,12 While vitronectin and collagen type I were
previously reported as MMP-2 and MMP-9 substrates,15 fibronectin has not been previously
recognized as an MMP-2 substrate. When we incubated p-aminophenylmercuric acetate
(APMA)-activated MMP-2, which is proteolytically active (Fig. 2A)16 with recombinant
fibronectin, we found cleavage of fibronectin by the protease. The finding that three important
ECM components are cleaved by MMP-2 and that MMP-2 increases adhesion raised the
possibility that cleavage of these ECM proteins was involved in cellular adhesion. To test this

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hypothesis, latent or APMA-activated MMP-2 was added to wells coated with vitronectin,
fibronectin or collagen type I for an hour (Fig. 2B). After addition of fluorescently-labeled
primary ovarian cancer cells, adhesion was determined using a fluorescence plate reader.
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Clearly, significantly more ovarian cancer cells adhered to the MMP-2-cleaved than to the full
length ECMs. Moreover, pre-incubation of the 3D model with activated MMP-2 also induced
adhesion. Conversely, pre-incubation of cancer cells with the various MMP-2-cleaved ECM
proteins reduced adhesion to the 3D model (Fig. 2C). In conjunction with our most recent12
and previous reports17-19 describing a requirement for α5β1-and αvβ3-integrin in ovarian cancer
cell adhesion, these results imply that pre-incubation of the ECM fragments with ovarian cancer
cells (Fig. 2C) occupies their respective integrin receptors which impairs the ability of the
cancer cell to adhere to the 3D model. Taken together, these data suggest that MMP-2 cleaves
vitronectin, fibronectin and collagen I and, thereby, increases ovarian cancer cell adhesion.

Activated MMP-2 increases early ovarian cancer invasion

Since adhesion and invasion are involved in the first steps of metastasis, we asked if
proteolytically cleaved ECM proteins also promote invasion. The three ECM proteins were
used to coat 35 mm glass bottom dishes and APMA or APMA-activated MMP-2 was added
to dishes for 1 hour. The well was washed and fluorescently-labeled SKOV3ip1ovarian cancer
cells added. 3D images were acquired in real-time using a Zeiss LSM510 confocal microscope
equipped with an environmental chamber (5% CO2, 37°C). For all three ECMs, cancer cells
invaded faster to a depth of 20 μM when the ECM was pre-incubated with proteolytically
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active-MMP-2 (Fig. 3 compare hatched to checkered areas). After quantifying the number of
invading cells with Image J (NIH) we found that MMP-2 not only facilitates adhesion but that
more cells invaded the ECM. When only APMA was present, only 96.4 +/- 10.2 cells bound
and invaded through undigested fibronectin, while an average of 141.3 +/- 11.1 cells bound
and invaded the MMP-2-digested ECM. Similar increases in cell numbers were found when
wells were coated with vitronectin (107.3 +/- 12.2 vs. 188.1 +/- 16.8 cells) or collagen I (54.2
+/- 7.1 vs. 80.3 +/- 6.2 cells). To confirm that proteolytically active MMP-2 is also important
when other stromal cells are present, we performed live in vitro imaging of the organotypic
3D culture after incubation with latent APMA alone or APMA-activated MMP-2 (Fig. 4A).
After addition of APMA or APMA-activated MMP-2 to the organotypic 3D culture for 1 hour
the wells were washed and fluorescently-labeled ovarian cancer cells were added in serum-
free media and images acquired in real-time (Fig. 4A). Addition of proteolytically active-
MMP-2 significantly increased ovarian cancer cell adhesion and invasion into the 3D culture
over an imaging time of 40 minutes. When APMA was present, only 102.6 +/- 7.4 cells bound
and invaded through 3D omental culture, while in the presence of APMA-activated MMP-2
an average of 179.4 +/- 16.3 cells bound and invaded the culture. In the opposite experiment,
MMP-2 or MMP-9 was downregulated in the cancer cells using a siRNA (Fig. 4B). The 3D
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culture was assembled and the siRNA-transfected ovarian cancer cells fluorescently-labeled
and imaged in real-time as they adhered and invaded the 3D model. MMP-2 deficient ovarian
cancer cells were unable to adhere and invade efficiently. Knocking down MMP-9 did not
affect the ability of SKOV3ip1 cells to bind and invade the 3D culture.

Early inhibition of MMP-2 reduces in vivo intraperitoneal metastasis and tumor load
These data suggest that MMP-2 is important for early adhesion and invasion. To determine if
MMP-2 plays a role on in vivo tumorigenesis, we transiently knocked down MMP-2 or MMP-9
in ovarian cancer cells by transfecting MMP-2 or MMP-9 specific siRNAs and injecting the
cells into the peritoneal cavity of athymic nude mice (Fig. 5). In two different ovarian cancer
cell lines, CaOV3 and SKOV3ip1, inhibition of MMP-2 reduced the number of metastases 4–
6 weeks later by an average of 43% and 47% (Fig. 5A), respectively, and the tumor weight by
an average of 36% and 49% (Fig. 5B), respectively, compared to the MMP-9 treated cancer
cells. These results substantiated previous results where we either pre-treated SKOV3ip1 or

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Hey A8 ovarian cancer cell lines one time with an inhibitor or antibodies against MMP-2 and
MMP-9, or performed repeated treatments (n = 12 treatments) with the inhibitor or antibodies
after tumors had established (2 weeks after intraperitoneal injection). Four weeks after
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intraperitoneal injection of tumor cells animals that received repeated treatments with the MMP
inhibitors had significantly more metastases and a greater tumor burden than the animals that
had received a single treatment at the beginning of the experiment.12

The role of MMP-2 in ovarian cancer metastasis

For a long time it was thought that the role of MMP-2 was to degrade the ECM so that cancer
cells could migrate through breaks in the basement membrane.20 Recent studies, however, have
indicated a more complex role for MMP-2 in cancer progression, since MMP-2 can release
growth factors from the ECM and regulate angiogenesis.21,22 The results presented here, in
conjunction with our previous studies,9,12,16 add yet another function to MMP-2. Immediately
after attachment to human peritoneum or omentum tumor cells upregulate MMP-2
transcriptionally,12 which leads to simultaneous cleavage of several ECM proteins on the
surface of the peritoneal cavity. These smaller fragments provide a scaffold for anchoring the
ovarian cancer cell to the surface and promote cancer cell invasion. These results are supported
by a report on the role of MMP-2/9 in the RIP1-Tag2 model of pancreatic islet carcinogenesis.
22 Treatment of early neoplasia with the MMP-2/9 inhibitor Batimastat resulted in significantly
smaller tumors, while repeated treatment of advanced islet adenocarcinomas with Batimastat
had no effect on tumor growth and weight.
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Ovarian cancer cells metastasize quickly

The current working model for the metastatic cascade in ovarian carcinoma suggests that cancer
cells are shed from the ovarian tumor into the peritoneal cavity and attach to the layer of
mesothelial cells that line the surface of the peritoneum and omentum, but often don't invade
very deeply into ECM (Fig. 1). Based on the results presented here, in the second part of the
metastatic process, attachment and invasion at the secondary site, is a very fast process as after
only 4 hours ovarian cancer cells can invade to a depth of 40 μM below the mesothelial cell
surface (Fig. 4). These observations were confirmed when we performed in vivo short term
adhesion assays by injecting ovarian cancer cells into the abdominal cavity of nude mice.
Within 24 hours cancer cells had attached firmly to the abdominal peritoneum and a significant
percentage homed to the omentum (data not shown). These results clearly show that ovarian
cancer is a fast metastasizing tumor that, once detached, is highly efficient in seeding within
the peritoneal cavity. These findings have potential clinical implications since they suggest
that the only time one can truly assume an ovarian cancer is localized to the ovary is when it
grows inside the ovary or in an ovarian cyst “protected” by a benign/stromal capsule (FIGO
Stage IA or IB tumors limited to one or both ovaries without cancer on the ovarian surface).
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For all other disease stages, such as stage IC, which is characterized by a tumor that has reached
the ovarian surface, or stage IIA-C tumors which are “limited” to the pelvis, one should
probably assume that ovarian cancer cells have metastasized microscopically throughout the
abdominal cavity and treat the patient accordingly. This hypothesis is supported by several
studies showing that patients with early stage ovarian cancer (stage I) benefit from adjuvant
chemotherapy after surgery (reviewed in ref. 23).

In summary, MMP-2 functions as an early response protein in ovarian cancer cells, facilitating
and accelerating their initial metastasis. MMP-2 cleaves ECM proteins into smaller fragments
which are then bound by integrins expressed on the ovarian cancer cell surface, allowing rapid
adhesion and invasion. These findings raise the possibility that the early inhibition of MMP-2
proteolytic activity or expression may effectively delay ovarian cancer metastasis.

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Acknowledgments
We are very grateful to Gail Isenberg for critical review of the manuscript. We are grateful to Dr. William Stetler-
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Stevenson, National Cancer Institute, for the human recombinant MMP-2.

Hilary Kenny, Ph.D. was supported by a Graduate Training Program in Cancer Biology Postdoctoral Fellowship
through the University of Chicago (NIH/NCI 5T32 CA09594).

Ernst Lengyel, M.D., Ph.D. holds a Clinical Scientist Award in Translational Research from the Burroughs Wellcome
Fund and is supported by grants from the Ovarian Cancer Research Fund (Liz Tilberis Scholars Program) and the NCI
(RO1 CA111882).

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Abbreviations
MMP matrix metalloproteinase
ECM extra-cellular matrix
3D three-dimensional
FN fibronectin
Vn vitronectin
Col I collagen type I
APMA p-aminophenylmercuric acetate
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Figure 1.
Histology of omental metastasis of human ovarian cancer. Hematoxylin and eosin stain of (A)
normal human omentum (MC, mesothelial cells, BM, basement membrane, Fib, fibroblasts,
Adi, adipocytes) and (B and C), ovarian cancer omental metastasis (Met, ovarian cancer
metastasis). Experimental details were as described in ref.9 Bar in bottom right of each panel
= 100 μm in length.
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Figure 2.
MMP-2 cleavage of vitronectin, fibronectin, collagen I increases primary ovarian cancer cell
adhesion. (A) Zymogram. MMP-2 was activated by APMA for 1 hour and was subjected to
gelatin zymography.12 (B) Adhesion assay. Full-length Vn (vitronectin), FN (fibronectin) and
Col I (collagen I) or the 3D omental model were plated in a 96-well dish. MMP-2 or MMP-2
that was proteolytically activated by APMA was added to the indicated ECM or the 3D model
(Cleavage of ECMs was confirmed by silver staining—not shown). After an 1 hour incubation,
the wells were washed with serum-free media and fluorescently-labeled primary human
ovarian cancer cells were added for 30 min. The wells were washed, fixed and fluorescence
measured using a fluorescent plate reader. (B) Competition assay. Primary human ovarian

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cancer cells were pre-incubated with MMP-2/ECM or APMA-activated MMP-2/ECM for 30

min. in serum-free media. Then the cells were added to the 3D omental model and adhesion
quantified as described in (A). *p-value <0.01, **p-value <0.001.
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Figure 3.
Activated-MMP-2 increases early ovarian cancer invasion through fibronectin, collagen I and
vitronectin. Live in vitro imaging of SKOV3ip1 cell adhesion and invasion into fibronectin,
collagen I and vitronectin. 200 μg of ECM was plated in a 35 mm glass bottom dish, APMA
or APMA-activated MMP-2 added for 1 hour, and the well washed with serum-free media.
Fluorescently-labeled SKOV3ip1 cells were added and real-time confocal microscopy images
taken at 0 μm depth which represents the surface of ECM and a 20 μm depth below the ECM
surface. Representative images at 2, 20 and 40 min. are shown.

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Figure 4.
MMP-2 increases early ovarian cancer invasion into the 3D omental model. Live in vitro
imaging of SKOV3ip1 cell adhesion and invasion into the 3D model. (A) After coating the 35
mm glass bottom dish with 100 μg type I collagen, 24,000 primary human fibroblasts were
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mixed with collagen and plated. Then a single, confluent layer of primary human mesothelial
cells was plated on top. APMA or APMA-activated MMP-2 was added and digestion occurred
for 1 hour. The dishes were washed with serum-free media and fluorescently-labeled
SKOV3ip1 cells were added. Real-time confocal microscopy images were obtained at 2, 20
and 40 min. and at 0 μm depth which represents the surface of the mesothelial cell layer and
at 40 μm depth below the surface of the 3D omental model. (B) SKOV3ip1 cells were
transfected with a siRNA specific for MMP-2 and MMP-9. 48 h post-transfection the
SKOV3ip1 cells were fluorescently-labeled and added to the 3D omental model. Real-time
confocal microscopy images were obtained at 0 μm depth which represents the surface of the
3D omental model and a 40 μm depth below the surface of the 3D omental model (mesothelial
cell layer) at the indicated times (2, 60, 120 and 240 min.).

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Figure 5.
Transient knock-down of MMP-2 significantly inhibits ovarian cancer metastasis. SKOV3ip1
or CaOV3 cells were transfected with siRNA specific for MMP-2 and MMP-9 or a control
siRNA. 48 h post-transfection 1 million SKOV3ip1 or 4 million CaOV3 cells were injected
intra-peritoneally into athymic nude mice. After 30 and 48 days, SKOV3ip1 and CaOV3
xenograft model tumors were collected, respectively. (A) The total number of metastasis and
(B) tumor weight was determined.12 *p < 0.01.
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