title: The role of the urokinase family in invasion by breast cancer
Creators: name: Qazi, Romena
Publisher: name: University of Glasgow
Year: 1998
Subjects: subject: Oncology.

description: Breast cancer is the leading cause of cancer mortality in women in the Western world. Its growth and development is directly governed by endogenous steroids and growth factors. Endocrine therapy is the most effective form of treatment for a proportion of breast cancer patients. Work from several laboratories has shown that breast cancer cells metastasise via a pathway that involves the plasminogen activator system and metalloproteinases. Thus, the plasminogen activator system, which consists of urokinase (uPA), its inhibitor (PAI-1) and receptor (uPAR), is central to the process of invasion and metastasis. Given their important role, it is hardly surprising that these three proteins have been used as biological markers of patient prognosis. uPA and PAI-1 has been significantly implicated as markers of aggressive breast cancer. The first step of invasion involves the conversion of plasminogen into plasmin by uPA. uPA is localised on the cell surface (bound to its receptor uPAR) and is principally regulated by two inhibitors, plasminogen activators type-1 and type-2 (PAI-1 and PAI-2). Possessing a wide substrate specificity, plasmin in turn activates a variety of enzymes which degrade different components of the extracellular matrix such as laminin, fibronectin, collagenase(s), proteoglycans etc. The present study was conducted to further understand the effect(s) of different growth factors and steroid hormones on the proliferation and invasive potential of two well characterised human breast cancer cell lines, MCF-7 (hormone sensitive) and MDA-MB-231 (hormone insensitive). Furthermore, extracellular matrix (ECM) was used to determine the effect, if any that support substrate might have on the levels of plasminogen activators and growth rate. Plasminogen activators were quantified both at the mRNA and protein levels, using RT-PCR and ELISA, respectively. uPA activity was determined by chromogenic assay and the invasive properties of the two cell lines were determined using the Boyden invasion chamber. In the initial phase, proliferation of each cell line on both substrata, plastic and EHS, was studied after treatment with growth factors (TGFalpha, TGFbeta and EGF), steroid hormones (E2 and Pg) and anti-oestrogen, tamoxifen. Different amounts of each growth factor and hormone were used and cell numbers were obtained after 24, 48 and 72 hours. These studies showed that growth of MCF-7 cells was stimulated significantly by exogenous TGFalpha and EGF, on both EHS and plastic with cells being more sensitive to growth factors on EHS. Proliferation of MDA cells was only slightly increased by the same growth factors on either substrate. By contrast, TGFbeta inhibited the growth of MDA cells, on both surfaces. However, the concentrations required for inhibition on the two substrata were enormously different. E2, Pg, and TAM (an anti-oestrogen) also stimulated the growth of MCF-7 cells on both plastic and EHS. For quantification of uPA, uPAR and PAI-1, a combination of Western blotting and ELISA, using specific monoclonal antibodies, was employed to monitor the relative amounts of these proteins after treating the cell lines with growth factors and hormones. These studies showed that the MDA cell line expressed uPA and PAI-1 proteins whereas uPA was hardly delectable in the MCF-7 cell line. Moreover, qualitative RT-PCR analysis clearly showed that uPAR and PAI-1 transcripts were present in both MDA and MCF-7 cells. These results clearly show that various growth factors and/or hormones not only have an effect on the proliferative properties of cell lines but they also change the relative amounts of uPA, PAI-1 and uPAR and that these changes can be reflected in invasive activity. The ECM contributes to the overall response of these cells. (Abstract shortened by ProQuest.).