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Ovarian cancer is an highly metastatic disease characterized by ascites formation and diffuse i.p. adhesion, invasion, and metastasis. Levels of lysophosphatidic acid (LPA) are elevated in the plasma of patients with ovarian carcinoma, including 90% of patients with stage I disease, suggesting that LPA may promote early events in ovarian carcinoma dissemination. Expression of matrix metalloproteinases (MMPs) is also up-regulated in ovarian cancer tissues and ascites, and numerous studies have provided evidence for a direct role of MMPs in i.p. invasion and metastasis. Using three-dimensional type I collagen cultures or immobilized beta1 integrin subunit-specific antibodies, we previously demonstrated that beta1 integrin clustering promotes activation of proMMP-2 and processing of membrane type 1 MMP in ovarian cancer cells (S. M. Ellerbroek et al., Cancer Res., 59: 1635-1641, 1999). In the current study, the effect of LPA on MMP expression and invasive activity was investigated. Treatment of ovarian cancer cells with pathophysiological levels of LPA increased cellular adhesion to type I collagen and beta1 integrin expression. A significant up-regulation of MMP-dependent proMMP-2 activation was observed in LPA-treated cells, leading to enhanced pericellular MMP activity. As a result of increased MMP activity, haptotactic and chemotactic motility, in vitro wound closure, and invasion of a synthetic basement membrane were enhanced. These data indicate that LPA contributes to metastatic dissemination of ovarian cancer cells via up-regulation of MMP activity and subsequent downstream changes in MMP-dependent migratory and invasive behavior.

Activation of the epidermal growth factor (EGF) receptor regulates many processes associated with metastasis, including modulation of cell:cell and cell:substrate interactions, production of matrix-degrading proteinases, and cellular migration. We have demonstrated previously that EGF stimulates migration and matrix metalloproteinase (MMP)-9-dependent invasion of ovarian cancer cells. In this study, we compare the roles of EGF-induced phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) activities in regulation of cellular responses associated with ovarian tumor cell metastasis. Inhibition of PI3K and MAPK activity impairs EGF-stimulated cell migration, in vitro invasion, and MMP-9 production. PI3K activity is not required for growth factor disruption of cell:cell junctions, whereas inhibitors of extracellular signal-regulated kinase (ERK)1/ERK2 activation and p38 MAPK activity block EGF-dependent junction dissolution. EGF promotes pro-MMP-9 binding to the cell surface through a mechanism that is independent of extracellular enzyme concentration. Interestingly, inhibition of PI3K activity abolishes EGF-induced cell surface association of pro-MMP-9, whereas inhibitors of MAPKs only partially block the response. These data suggest that EGF receptor activation promotes a PI3K-dependent induction of a cell surface pro-MMP-9 binding component that may facilitate gelatinase-mediated cellular invasion and supports an expanded role for elevated PI3K activity in cellular responses associated with ovarian tumor metastasis. In addition, our findings support the hypothesis that divergent kinase activities regulate distinct cellular events associated with growth factor-induced invasion of ovarian cancer cells.

Hematogenous metastasis is postulated to involve tumor cell-initiated degradation of basement membrane barriers and underlying connective tissue matrices. Matrix metalloproteinases (MMP) are zinc-dependent endopeptidases that have been implicated in the proteolytic events of tumor cell invasion. Research has revealed a class of membrane-anchored metalloproteinases (MT-MMPs) and has provided convincing evidence that these enzymes activate latent MMP-2 (72 kDa gelatinase A) on the cell surface. The activation of plasma membrane associated MMP is a potential mechanism for facilitation of cellular metastasis and requires consideration when addressing potential roles of MMPs in tumor progression. This review focuses on potential in vivo regulatory mechanisms of membrane-associated MMP activity in the context of tumor cell interaction with matrix macromolecules. BioEssays 1999;21:940-949.

Culturing DOV 13 ovarian carcinoma cells on three-dimensional collagen lattice but not on thin-layer collagen induces processing of promatrix metalloproteinase (MMP)-2 to a M(r) 62,000 form, suggesting that multivalent integrin aggregation may participate in proteinase regulation. To address the role of collagen-binding integrins in this event, we treated DOV 13 cells with soluble beta1 integrin antibodies (clones P4C10 or 21C8) or beta1 integrin antibodies immobilized on latex beads to promote integrin aggregation. Divalent ligation of beta1 integrins with soluble P4C10 antibodies stimulated expression of pro-MMP-2 and its inhibitor, tissue inhibitor of metalloproteinase-2, whereas soluble 21C8 antibodies had no effect. Aggregation of beta1 integrins with immobilized 21C8 or P4C10 antibodies stimulated MMP-dependent pro-MMP-2 activation and accumulation of a M(r) 43,000 form of membrane type 1 MMP (MT1-MMP), a cell surface activator of pro-MMP-2, in cell extracts. beta1 integrin-mediated MMP-2 activation required protein synthesis and tyrosine kinase signaling and was reduced by an inhibitor of gene transcription. Treatment of control cells with concanavalin A stimulated MMP-dependent pro-MMP-2 activation and accumulation of M(r) 55,000 and 43,000 forms of MT1-MMP in cell extracts. Addition of either the MMP inhibitor GM-6001-X or exogenous tissue inhibitor of metalloproteinase-2 to concanavalin A-treated cells resulted in loss of the M(r) 43,000 form of MT1-MMP and accumulation of the M(r) 55,000 form of the enzyme in cell extracts, suggesting that the M(r) 43,000 form is a product of MMP-dependent M(r) 55,000 MT1-MMP proteolysis. Together, these data suggest that beta1 integrin stimulation of pro-MMP-2 activation involves MT1-MMP posttranslational processing and requires multivalent integrin aggregation.

Aberrant expression or activity of the epidermal growth factor (EGF) receptor family of tyrosine kinases has been associated with tumor progression and an invasive phenotype. In this study, we utilized 4 ovarian cancer cell lines, OVCA 432, DOV 13, OVEA6 and OVCA 429, to determine the effects of EGF on the regulation of proteolytic enzymes and their inhibitors, cellular migration and in vitro invasion. Induction of urinary-type plasminogen activator (u-PA) activity and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was observed in all 4 cell lines. OVCA 432 cells showed strong PAI-1 induction; however, the other 3 lines displayed substantial baseline PAI-1 expression that was not induced by EGF. EGF-dependent stimulation of migration and induction of matrix metalloproteinase (MMP)-9 (gelatinase B) was observed in OVEA6 and OVCA 429 cells only. Upon EGF receptor activation, DOV 13, OVEA6 and OVCA 429 cells were induced to invade through an artificial basement membrane (Matrigel); however, no invasion was detected in OVCA 432 cells. Cell lines displaying induction of migration and MMP-9 (OVEA6 and OVCA 429) demonstrated robust EGF-induced invasion (5- to 20-fold), and cell invasion was substantially reduced in the presence of anti-catalytic MMP-9 antibody. Addition of anti-catalytic u-PA antibody inhibited the modest (2-fold) EGF-induced invasion in a cell line that did not express MMP-9 (DOV 13) and in OVEA6 cells that displayed the highest baseline u-PA activity. Together, our findings indicate that multiple proteinases are important in ovarian cell invasion and implicate EGF induction of MMP-9 and migration as key components of more aggressive ligand-induced invasion.