Angiogenesis is the rate-limiting step in tumour progression and metastatic spread. Both promoters and inhibitors of angiogenesis have been implicated in thyroid tumourigenesis. We have reported PTTG overexpression in thyroid and other cancers, and PTTG upregulation of the angiogenic factors FGF-2 and VEGF in vitro. To investigate whether PTTG also transactivates other downstream angiogenic effectors, we employed angiogenesis-specific cDNA arrays. Primary thyroid cells and PTTG-null HCT113 cells were transiently transfected with wild-type (WT)-PTTG and empty vector. After mRNA and protein verification, angiogenic factors which demonstrated more than 2-fold changes in expression averaged over 3 consecutive cDNA arrays were selected for further investigation. Thrombospondin-1 (TSP-1), a secreted anti-angiogenic factor, showed a mean 2.7-fold downregulation in response to PTTG overexpression. Conversely, the pro-angiogenic factor ID3 (inhibitor of DNA-binding-3) showed a 3.8-fold induction. We subsequently validated these candidate genes using TaqMan RT-PCR. PTTG downregulated TSP-1 86 % (N = 5, P<0.001) in primary thyroid cells and 73 % (N = 3, P<0.05) in HCT113 cells compared to vector-only cells. By contrast, PTTG upregulated ID3 62% in primary thyroid cells (N = 3, P = 0.005), and transfection studies in FTC133 thyroid follicular cells further supported PTTG upregulation of ID3 (4.35-fold induction, N = 3, P < 001). In addition, we validated our findings at the protein level. We next examined TSP-1 and ID3 expression in our thyroid cancer cohort. In keeping with our in vitro data, TSP-1 was reduced in cancers (N = 26) compared to normal thyroids (N = 6). ID3, however, was significantly raised in tumours compared to normal tissue (5.57-fold, P = 0.025). We conclude, therefore, that PTTG's promotion of angiogenesis in vivo may be via altered regulation of several genes with both pro- and anti-angiogenic properties, and that we have identified ID3 and thrombospondin-1 as two new downstream targets of PTTG.
22 - 24 Mar 2004
British Endocrine Societies