A critical role for the proto-oncogene PBF in regulating cellular adhesion and motility in thyroid cancer

The progression of thyroid cancer is dependent on cell motility, a highly complex process that involves the co-ordination of multiple signalling pathways, cell adhesion, and actin dynamics. The proto-oncogene pituitary tumor-transforming gene (PTTG)-binding factor (PBF/PTTG1IP) potently stimulates thyroid cancer cell migration and invasion via PBF phosphorylation by Src kinase at residue Y174. Recent phosphoproteomic and RNA-Seq analyses revealed that upregulation of PBF in Nthy-ori thyroid cells resulted in altered expression and phosphorylation of adhesion and cytoskeletal proteins. We thus hypothesized that PBF is a physiological regulator of cell adhesion and its overexpression in tumours promotes cell motility via altered adhesion. Our preliminary studies have utilized TPC-1 human papillary thyroid carcinoma cells with CRISPR/Cas9-mediated PBF knockout (KO) in the analysis of cell adhesion and spreading on fibronectin-coated plates. Cell adhesion assays demonstrated that PBF-depleted cells exhibited markedly decreased cell-substrate adhesion at multiple time points up to 4 hours compared with controls. We then assessed focal adhesions, the large protein complexes that link the cell cytoskeleton to the extracellular matrix. Immunofluorescence staining of focal adhesion kinase (FAK) and paxillin revealed fewer focal adhesions with altered distribution in TPC-1 PBF KO cells compared with control cells. In particular, FAK and paxillin stained structures were smaller and shorter with reduced frequency in PBF KO cells vs controls, which displayed numerous, elongated focal adhesion structures along actin fibres. Initial live cell imaging of LifeAct-GFP and cell spreading assays also suggested that PBF KO cells had impaired cell spreading. Conversely, exogenous PBF expression resulted in a greater number of dense paxillin structures which were stabilized at focal adhesions in the lamellipodia. Altogether, these findings provide new important insights into focal adhesion dynamics in PBF-induced thyroid cancer cell motility. Further investigations are now required to better define the precise PBF interactions with cell adhesion protein complexes.