Year

2007

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences - Faculty of Science

Abstract

Cellular invasion is facilitated by localisation and activation of the human circulating zymogen plasminogen. Increased binding and activation of plasminogen at the surface of invasive breast cancer cells has been linked to the inappropriate and unregulated expression of the plasminogen activator, uPA. To investigate the mechanistic link between uPA and plasminogen binding, cell-surface uPA levels were enhanced on the usually low uPA-expressing MCF-7 cell line. Enhancement of cell-surface uPA resulted in a significant increase to the cell-surface, lysine-dependent plasminogen binding capacity of these cells (increase of 37%). This increase was entirely uPA activity-dependent and was accompanied by an increase in plasminogen activation potential. Analysis in the presence of the plasmin inhibitor, aprotinin, showed that a large proportion of plasminogen binding was also plasmin-dependent (70-80%). Similar analyses indicated that this was also the case on MDA-MB-231 cells, which have naturally high levels of cell-surface uPA. Investigation into the identity of potential cell-surface plasminogen receptors showed that a proportion of cell-surface, lysine-dependent plasminogen binding to MDA-MB-231 cells was mediated via a direct interaction with the A-chain of uPA. Furthermore, fluorescent confocal microscopy experiments indicated that plasmin-processed annexin II may act as a plasminogen receptor on uPA-enhanced MCF-7. Together with the results above, these data suggest a mechanism whereby a low level of plasminogen may initially localise to receptors with pre-existing C-terminal lysines and, on cells with high cell-surface uPA, a feed-forward activation loop proceeds. This activation loop leads to generation of plasmin and subsequent plasmin-processing of cell-surface proteins, thereby generating new C-terminal lysines that facilitate further lysine-dependent plasminogen binding and activation. Primary tumour expression of uPA and its inhibitor, PAI-1, is a strong prognostic indicator of adverse patient outcome in breast cancer, and uPA expression is also associated with malignant disease in other cancers. Radiolabelled PAI-2 (α-PAI-2) is a promising therapeutic which specifically targets uPA-expressing cells, however, pre-clinical evaluation of α-PAI-2 toxicity and efficacy requires an appropriate mouse model of breast cancer. To this end, immunocompromised mice bearing orthotopic MDA-MB-231 xenografts were assessed as a suitable model for invasive breast cancer. The model was characterised for tumour induction and growth rate, necrosis, local invasion into surrounding tissue, and metastases to axillary lymph nodes and major organs. Tumours were also characterised for uPA expression (using an ELISA developed as part of this thesis), uPA activity and plasminogen activation capacity, both with and without α-PAI-2 treatment. These analyses indicated that the orthotopic murine xenograft model was appropriate for assessing α-PAI-2 efficacy, as the xenogenic tumours maintained the characteristics of the implanted cell line and mimicked the development and histology of human breast tumours, including uPA expression by tumour stromal cells. The plasminogen binding model described in this thesis provides a plausible explanation for the increased plasminogen binding capacity of cells with high cell-surface uPA expression and furthers understanding of the link between increased uPA expression and increased tumour cell invasiveness. Furthermore, the characterisation of the xenograft mouse model highlights the suitability of this model for the pre-clinical investigation of potential uPA-targeted therapeutics for invasive breast cancer.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.