The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin
Hilary A. Kenny, … , Lisa M. Coussens, Ernst Lengyel
Hilary A. Kenny, … , Lisa M. Coussens, Ernst Lengyel
Published March 13, 2008
Citation Information: J Clin Invest. 2008;118(4):1367-1379. https://doi.org/10.1172/JCI33775.
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Research Article Oncology

The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin

Abstract

Most patients (80%) with ovarian cancer (OvCa) present with metastatic disease. Attachment of OvCa cells to peritoneum and omentum represents the first rate-limiting step for metastatic spread. Therefore, identifying factors regulating cell attachment in the abdominal cavity is critical to the development of therapeutic agents. We show here that MMP-2 expression was upregulated in OvCa cells upon attachment to their microenvironment. Downregulation of MMP-2 mRNA or pharmacological inhibition of MMP-2 proteolytic function, in both human OvCa primary cells and cell lines, reduced attachment of OvCa cells to a 3D organotypic model of metastatic OvCa, full human omentum or peritoneum, and in vivo to mouse peritoneum and omentum. Absence of MMP-2 in the host did not alter OvCa adhesion, as determined utilizing mice harboring homozygous null mutations in either the Mmp2 or Mmp9 genes. Conversely, adhesion induced upregulation of MMP-2 mRNA in OvCa cells. MMP-2 inhibition in OvCa cells through pharmacological or antibody treatment prior to i.p. dissemination in nude mice significantly decreased tumor growth and metastasis and extended survival. MMP-2 enhanced peritoneal adhesion of OvCa cells through cleavage of ECM proteins fibronectin (FN) and vitronectin (Vn) into small fragments and increased binding of OvCa cells to these FN and Vn fragments and their receptors, α5β1 and αVβ3 integrin. These findings indicate that MMP-2 expressed by metastatic OvCa cells functionally regulates their attachment to peritoneal surfaces.

Authors

Hilary A. Kenny, Swayamjot Kaur, Lisa M. Coussens, Ernst Lengyel

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Figure 7

MMP-2 cleavage of Vn and FN increases OvCa adhesion.

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MMP-2 cleavage of Vn and FN increases OvCa adhesion. (A) Human omentum a...
(A) Human omentum and peritoneum stained with Vn- or FN-specific antibodies (left panel). HPMCs were subjected to immunoblotting (right panel) using Vn- and FN-specific antibodies. Lane 1, HPMCs. HT-1080 CM was used as positive control. Original magnification, ×400. (B) Full-length FN was incubated with activated MMP-2, and fragments were resolved on a 10% Tris-HCl gel by silver stain analysis. (C) Adhesion assay of SKOV3ip1 cells to full-length and MMP-2–cleaved Vn or FN coated plates as described in Figure 2. (D) FN and MMP-2–cleaved FN were run on a native gel and transferred to nitrocellulose. An adhesion assay was performed with 1.0 × 107 SKOV3ip1 cells for 4 hours, the membrane was washed, fixed, and bound cells were stained. (E) Competition assays were conducted. SKOV3ip1 cells were preincubated with full-length Vn, MMP-2–cleaved Vn, full-length FN, or MMP-2–cleaved FN, and an adhesion assay to 3D coculture was conducted. (F) SKOV3ip1 cells were transfected with a siRNA specific for α5 or β3 integrin, and adhesion assay was performed to MMP-2–cleaved Vn or FN coated plates. (G) Fluorescently labeled SKOV3ip1 cells were pretreated with either an MMP-2 or a mouse isotype IgG antibody followed by treatment with α5, β1, αvβ3, β4 integrin, or mouse isotype IgG antibodies. Subsequently, an adhesion assay was performed on the 3D model. *P < 0.01, **P < 0.001. Each graph and picture is representative of 3 independent experiments. Scale bar: 100 μm.