Pituitary Tumor Transforming Gene (PTTG) Regulates Downstream Angiogenic Genes in Thyroid

Problem: Angiogenesis is the rate-limiting step in tumor progression and metastatic spread. Both promoters and inhibitors of angiogenesis have been implicated in thyroid tumorigenesis. We have reported PTTG overexpression in thyroid cancer, and PTTG upregulation of the angiogenic factors FGF-2 and VEGF. This study was conducted to investigate whether PTTG also transactivates other downstream angiogenic effectors. Methods: To investigate whether PTTG also transactivates other downstream angiogenic effectors, we used angiogenesis-specific cDNA arrays. Primary thyroid cells and PTTG-null HCT113 cells were transiently transfected with wild-type-PTTG. Angiogenic factors that demonstrated more than 2-fold changes in expression averaged over 3 cDNA consecutive arrays were selected for further investigation. Results: Thrombospondin-1 (TSP-1), an anti-angiogenic factor, showed a mean 2.7-fold downregulation in response to PTTG overexpression. Conversely, the pro-angiogenic factor ID3 showed a 3.8-fold induction. We subsequently validated these candidate genes using TaqMan RT-PCR. PTTG downregulated TSP-1 86% (P < 0.001) in primary thyroid cells and 73% (P < 0.05) in HCT113 cells compared to vector-only cells. By contrast, PTTG upregulated ID3 62% in primary thyroid cells (P = 0.0053), and transfection studies in FTC133 thyroid follicular cells further supported PTTG upregulation of ID3 (4.35-fold, P < 0.01). In addition, we validated our findings at the protein level. We next examined expression in our thyroid cancer cohort. In keeping with our in vitro data, TSP-1 was reduced in cancers compared to normal thyroids. ID3, however, was significantly raised in tumors compared to normal tissue (5.57 fold, P = 0.025). Conclusion: We conclude that PTTG promotion of angiogenesis may be via altered regulation of several genes with both pro-(ID3) and anti-angiogenic (TSP-1) properties. Significance: We have identified PTTG, ID3, and thrombospondin as potentially important anti-angiogenic targets. It is hoped that this will improve the future treatment of the more aggressive thyroid cancers that are associated with very poor prognosis. Support: None reported.