The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin
Hilary A. Kenny, Swayamjot Kaur, Lisa M. Coussens, Ernst Lengyel
Hilary A. Kenny, Swayamjot Kaur, Lisa M. Coussens, Ernst Lengyel
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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 3

Attachment of OvCa cells depends on MMP-2.

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Attachment of OvCa cells depends on MMP-2. (A) SKOV3ip1 cells were trans...
(A) SKOV3ip1 cells were transfected with a siRNA specific for MMP-2, MMP-9, or a control siRNA and plated on collagen type I, and MMP-2 and MMP-9 were detected by immunoblotting with specific antibodies. The membrane was reprobed with actin. (B–D) Adhesion assays. SKOV3ip1 cells were transfected with a siRNA specific for MMP-2 and MMP-9. SKOV3ip1, HeyA8, and primary OvCa cells were pretreated with an MMP-2– or MMP-9–blocking antibody and then fluorescently labeled. OvCa cells were added to the 3D culture (B), a piece of full human omentum (C), or full human peritoneum (D), and the adherent cells quantified with a fluorescence reader as described in Figure 2. (E) SKOV3ip1 cells (1 × 106) were labeled fluorescently and injected i.p. into nude mice for 4 hours (n = 3). Mice were sacrificed after 4 hours, omentum and peritoneum was lysed with NP-40, and fluorescence was measured. (F) Adhesion assay. SKOV3ip1 cells (5 × 104) pretreated with the MT1-MMP antibody were added to the 3D culture and an adhesion assay performed as described in Figure 2 (left panel). Invasion assay (right panel). SKOV3ip1 cells (3 × 104) were treated with a MT1-MMP– or MMP-9–blocking antibody, and invasion was analyzed after 24 hours. *P < 0.01.