Elucidating the mechanisms via which the proto-oncogene PBF stimulates cell motility in thyroid tumour progression

Background and Aims: The proto-oncogene pituitary tumor-transforming gene binding factor (PBF/PTTG1IP) is overexpressed in multiple tumours, including thyroid cancer, and is associated with tumour progression. In vitro, PBF potently induces cancer cell motility, and both Src-mediated phosphorylation at tyrosine 174 (Y174) and endocytosis of PBF are required for induction of thyroid and breast cancer cell migration and invasion. This study aimed to further elucidate the mechanisms by which PBF induces cancer cell motility.Methods: To elucidate molecular events downstream of PBF overexpression, phosphoproteomic and RNA-Seq analyses of thyroid cells stably overexpressing PBF were performed. We then utilised a novel Pbf knockout (Pbf-/-) mouse model with CRISPR/Cas9-mediated deletion of Pbf exon 4 in C57BL/6N mice. Mouse embryonic fibroblasts (MEFs) were isolated at embryonic day 13.5 and used as primary cultures.Results: Phosphoproteomic and RNA-Seq analyses revealed enrichment for molecules involved in cell adhesion and cytoskeleton organisation in response to PBF overexpression, prompting further investigation into a physiological role for PBF in cell motility. Pbf-/- MEFs showed a significant reduction in migration and invasion compared with wild-type (Pbf+/+) MEFs. Interestingly, heterozygote MEFs (Pbf+/-) showed an intermediate decrease in motility suggesting a gene-dosage effect. Additionally, the migration of Pbf-/- MEFs transfected with PBF was comparable with Pbf+/+ MEFs in rescue experiments. Initial immunofluorescent studies of Pbf-/- MEFs suggest alterations in focal adhesion (FA) structure and distribution. Importantly, Pbf-/- MEFs demonstrated a marked reduction in focal adhesion kinase (FAK) and paxillin staining with smaller, punctate and more radially distributed FAs compared with Pbf+/+ MEFs.Conclusions: These findings further confirm a role for PBF in cell motility, through regulating cell adhesion and cytoskeleton organisation. Overall, these studies demonstrate a physiological role for PBF in cell motility and further elucidate the mechanisms by which PBF induces cell motility in thyroid tumour progression