PTTG Induces Genomic Instability in Thyroid Cells

3532 Cancer reflects the progressive accumulation of genetic alterations and subsequent genetic instability of cells. Cytogenetic studies have demonstrated the importance of aneuploidy in differentiated thyroid cancer development. Pituitary tumor transforming gene (PTTG), also known as securin, is a mitotic checkpoint protein which inhibits sister chromatid separation during mitosis. PTTG is highly expressed in many cancers and over-expression of PTTG induces aneuploidy in vitro. Using fluorescent inter-simple sequence repeat PCR and ABI377 sequencer we investigated the relationship between the degree of genetic instability and PTTG expression in normal and tumorous thyroid samples. The genomic instability index (GI-index) was 6.7-72.7% higher in cancers than normal, and in metastatic nodes from 2 patients were 104 & 225% greater than in the corresponding primary cancer. We also found that follicular thyroid tumors exhibited greater genetic instability than papillary tumors (27.6% (n = 9) versus 14.5% (n = 10), p = 0.03). By measuring PTTG mRNA using quantitative-PCR we demonstrated a relationship between PTTG expression and the degree of genetic instability in thyroid cancers (R2 = 0.80, p = 0.007). To further investigate PTTG’s role in genetic instability we transfected FTC133 thyroid follicular cells and observed increased genetic instability in cells over-expressing PTTG compared with vector-only controls (n = 3, GI-Index VO = 29.7 +/–5.2 vs. PTTG = 63.7 +/–6.4, p = 0.013). Consistent with the strong positive correlation in vivo, we also observed a dose response in genetic instability and PTTG expression (GI-Index low dose (0.5μg DNA/ 6-well plate) PTTG = 15.3% +/–1.7 vs. high dose (2 μg DNA) PTTG = 50.8% +/–3.3, p = 0.006). Confirming other reports, our Affymetrix array study of cellular response to irradiation showed significant downregulation of PTTG (2.3 fold) and a significant upregulation of Rad21 expression (1.9 fold). The potential relationship between the DNA repair gene rad21 and PTTG was further investigated in vivo and in vitro. In a cohort of matched normal and thyroid cancer specimens, PTTG mRNA expression negatively correlated with rad21 expression (R2 = 0.5, n = 7, p = 0.048). Transfection of FTC133 cells with PTTG elicited significant downregulation of rad21 mRNA expression (1.54 fold, n = 3, p = 0.009). Consistent findings were seen with HCT116 and H1299 cells. Overall, we describe the first use of FISSR-PCR in human cancers, and demonstrate that the degree of instability strongly correlated with PTTG-expression in differentiated thyroid cancer. Furthermore, in vitro studies demonstrated up-regulation of PTTG in thyroid cells may drive genetic instability through suppression of a rad21-dependent DNA repair mechanism. We conclude that PTTG may be an important gene in the mutator phenotype development in thyroid cancer.