Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
Breast cancer is a common but often aggressive disease. The initiator of coagulation, tissue factor (TF), is overexpressed in malignant breast tumor cells as well as in tumor specimen from breast cancer patients and may form a molecular marker for malignant breast cancer. However, next to upregulation on tumor cells, TF has also been shown to influence tumor growth. In colorectal cancer, tumor TF expression introduces a direct growth advantage for the tumor cells in vivo, and regulates tumor blood vessel formation (angiogenesis). TF also enhances tumor progression in pancreatic cancer models and interestingly, TF expression has recently been shown to enhance tumor growth in a breast cancer model. The mechanism by which TF regulates enhanced breast tumor growth is completely unknown, but TF-induced generation of downstream coagulation factors such as FVIIa and thrombin are thought to play a role. Coagulation factors induce activation of protease-activated receptors (PARs), leading to changes in cellular behaviour, such as cell growth and migration. In addition, PAR signalling, specifically FVIIa-induced PAR2 but not thrombin-induced PAR1 signalling, is regulated by the TF cytoplasmic tail; PAR2-dependent integrin function is inhibited by the TF cytoplasmic tail, whereas tail phosphorylation reverses this effect. We hypothesize that TF and FVIIa-induced PAR2, but not thrombin-induced PAR1 activation regulates breast cancer development by pathways that involve regulation of TF cytoplasmic tail function. To test this hypothesis, we propose the following strategies: 1) Breast cancer cells that tetracyclin-dependently express wild type TF, a TF mutant that allows FVIIa-induced PAR2 activation but not downstream coagulation factor-induced signalling, or a TF mutant that lacks the cytoplasmic tail, will be injected into the fatpad of immuno-deficient mice. Tumor growth of these cells in the presence and absence of tetracyclin will be monitored, and after sacrifice, histochemical analysis of primary and metastatic tumors will be performed, to check tumor cell and stromal cell proliferation rates, as well as a angiogenesis rates of the tumors. 2) To investigate the role of FVIIa and the TF cytoplasmic tail in integrin-dependent tumor cell behaviour, the breast cancer cells mentioned above will be subjected to migration assays, with or without FVIIa stimulation. A physical link between TF and integrins will also be established in cells grown on various integrin-activation substrates.