Enhanced Cell-Associated Fibrinolytic Pathway but Not Coagulation Pathway Activity Contributes to Motility in Metastatic Breast Cancer Cells.

Abstract 3170

Poster Board III-105

Activation of tumor cell-associated coagulation and fibrinolytic pathways occurs in many different malignant and metastatic disease processes, including breast cancer. It has been proposed that coagulation and fibrinolytic pathway activation highjack host hemostatic mechanisms and facilitate the metastatic process. However, depending on tumor cell gene expression, tumor microenvironment, and tissue of origin, it is not fully understood whether the coagulation pathway or fibrinolytic pathway, alone or together in concert, contributes to the metastatic phenotype. To characterize and compare the coagulation and fibrinolytic pathways of normal and metastatic cells, we utilized the MCF-10 family of breast cell lines. The MCF-10 family of breast cell lines was originally derived from a woman with benign fibrocystic breast disease, which led to the near normal immortalized MCF-10A cells. After transformation with T24-Ha-ras, more aggressive cell lines were derived, ultimately leading to the metastatic MCF-10CA1 cell line. Using an in vitro modified-Boyden chamber model, MCF-10CA1 cells were significantly more motile than MCF-10A cells both at baseline using BSA as the chemoattractant, and when using EGF as the chemoattractant. Thus, as predicted by properties previously reported in the literature, the metastatic MCF-10CA1 cells were substantially more motile than the near normal parental cell line, MCF-10A. Both cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. Using laser scanning confocal microscopy, we observed that MCF-10A cells produced a stable fibrin network, whereas MCF-10CA1 cells lysed the surrounding fibrin network within 24 hours of network formation. Importantly, fibrin located proximal to (within 10 microns of) the MCF-10CA1 cell surface lysed significantly faster than fibrin located 100 microns from the surface. These results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. A cell-surface plasminogen activation assay showed that MCF-10CA1 cells supported substantially increased plasmin generation rates compared to MCF-10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF-10CA1 cells expressed significantly increased levels of urokinase (uPA) and decreased levels of plasminogen activator inhibitor-1 (PAI-1) compared to the MCF-10A cells. Blocking uPA activity with an active-site directed-inhibitor (amiloride) decreased MCF-10CA1 cell motility to essentially the same level as MCF-10A cells. Inhibiting the phosphatidylinositol 3-kinase/Akt signaling axis of MCF-10CA1 cells with LY294002 similarly decreased cell surface plasminogen activation activity and cell motility. Collectively, these results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. Our results support continued investigation of urokinase inhibition, either by directly blocking uPA activity or by down-regulating uPA expression, as an attractive adjunctive therapeutic target to reduce metastatic breast cancer.