Elevated genetic instability is associated with repressed DNA repair gene expression in a bi-transgenic murine model of PTTG and PBF

Whilst the majority of differentiated thyroid cancers (DTC) have oncogenic mutations, a significant minority may be driven by the overexpression of proto-oncogenes. PTTG and PBF are proto-oncogenes which are induced in DTC, elicit tumours in xenograft models and interact in vitro, where PBF shuttles PTTG into the nucleus. However, the relative contributions of each gene to DTC has not been delineated. We constructed a bi-transgenic murine model over-expressing both PBF and PTTG specifically in the thyroid gland, and characterised it in comparison to age and sex matched single transgenic (PTTG-Tg, PBF-Tg) and wild-type (WT) mice. A total of 68 WT, 98 PBF-Tg, 25 PTTG-Tg and 23 bi-transgenic mice were assessed. No significant difference in thyroid weight was observed between male and female mice within each of the 4 genotypes. However, there was a significant 2.7-fold increase in thyroid weight in bi-transgenic mice compared to WT (p<0.001) at 6 weeks of age. PBF-Tg thyroid weight was 1.7-fold higher than WT mice (p<0.001), whereas PTTG thyroid weights were similar to WT (0.95-fold; p=0.18). Interestingly, bi-transgenic thyroids were 1.6-fold heavier than PBF-Tg thyroids (p<0.001). Enlarged thyroid growth in bitransgenic mice was accompanied by significant hyperplasia and macrofollicular lesions. As oncogenic expression of PTTG is known to induce genetic instability (GI), we determined GI levels through FISSR-PCR in primary thyroid cultures of each genotype. Compared with WT mice (arbitrary GI Index = 0%), PBF-Tg mice had a GI Index of 19.8±1.8%; PTTG-Tg mice of 7.6±1.6%; and bi-transgenic mice of 37.9±2.7%. Examination of a panel of 83 DNA repair genes to understand which genetic changes might underlie these differences showed expression of 9 genes were significantly down-regulated >1.5-fold in bi-transgenic thyrocytes compared to PBF-Tg, including Gadd45a (2.3-fold, p=0.006), Brca1 (2.1-fold, p=0.003) and Chek1 (2.4-fold, p=0.01). Together, our data reveal a complex interplay between PTTG and PBF in vivo; the bi-transgenic thyroid phenotype is closer to that of PBF-Tg mice than PTTG-Tg mice, but reveals increased goitre size, and heightened genetic instability than either single transgenic model alone.