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 1

Binding of OvCa cells to an omental 3D culture increases MMP-2/-9 expression and secretion in cancer cells.

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Binding of OvCa cells to an omental 3D culture increases MMP-2/-9 expres...
(A) Concept for a 3D culture to imitate abdominal mesothelium. HPMCs stained with an antibody against cytokeratin 8, vimentin, or an isotypic specific control antibody. HPFs stained with a prolyl-hydroxylase antibody recognizing human fibroblasts. Scale bar: 50 μM. (B) Zymogram. Conditioned media from SKOV3ip1 cells (red circles), the 3D culture (composed of HPMCs [blue rectangles], HPFs [yellow diamonds], and collagen I [green rectangles]), or coculture was subjected to gelatin zymography. (C) Gelatinase assay. Cell-associated gelatinolytic activity in the indicated cell populations, with or without an MMP-2/-9 inhibitor, was determined using a quenched fluorogenic peptide. Fluorescence was measured with a fluorescence spectrophotometer. (D) Schematic of adhesion assay and subsequent cell sorting. Fluorescently labeled SKOV3ip1 cells were mixed with 3D culture for 4 h. The cells were mechanically removed from the plate and sorted by FACS. (E) Cell extracts were subjected to immunoblotting using an MMP-2 and MMP-9 specific antibody. The membrane was reprobed with an antibody against actin. Lane 1, unbound, floating SKOV3ip1 cells; lane 2, SKOV3ip1 cells that had attached to the 3D culture after FACS; Lane 3, 3D culture alone; Lane 4, 3D culture that had bound SKOV3ip1 cells after FACS. (F) Western blot for MT1-MMP and TIMP-2. SKOV3ip1 were plated on the 3D culture and then sorted as indicated in D. Cell extracts (top panel) or conditioned media (bottom panel) were subjected to immunoblotting with MT1-MMP or TIMP-2 antibody. HT-1080 CM was used as positive control.