Endocrine Abstracts

Metastasis is a multistep process responsible for the vast majority of endocrine cancer deaths. We have previously identified the proto-oncogene PBF to be upregulated in differentiated thyroid cancer, and recently PBF expression has been correlated with distant thyroid cancer metastasis at diagnosis. Further, PBF potently induces breast cancer cell invasion in vitro, and our recent in vivo data demonstrate that colorectal tumours with higher PBF protein expression demonstrate increased vascular invasion. We now show that PBF significantly promotes cell invasion in thyroid carcinoma SW1736 cells compared to vector only (VO) (P<0.01). Cell invasion is related to and encompasses cell migration. In keeping with PBF’s role in cell invasion, overexpression of PBF in HCT116 cells induced ~60% greater migration compared to VO-transfected controls (P<0.001), implying a role for PBF in promoting cell movement. These data were supported by wound healing assays in MDA-MB-231 cells, which revealed that GFP-tagged PBF cells migrate significantly further than GFP-VO cells (VO=115.3 μm, PBF=143.0 μm, P<0.01). To begin to unravel the mechanism by which PBF promotes cell migration and invasion we used an IP-MS approach to discover PBF binding partners, and identified the cortical actin binding protein, cortactin. Interaction was confirmed through co-IP, immunfluorescence and proximity ligation assays. The latter additionally indicated that this interaction occurs within or close to the plasma membrane. PBF is phosphorylated by SRC at tyrosine 174 (Y174) and when phosphorylated it is preferentially located at the plasma membrane. PLA assays using the SRC inhibitor PP1 and co-IP experiments using a PBF phospho null mutant (Y174A) demonstrated that loss of PBF phosphorylation at Y174 abrogates the interaction between PBF and cortactin, suggesting that cortactin preferentially binds to phosphorylated PBF. We therefore examined the ability of the phosphorylation status of PBF to modulate its induction of cell invasion. Substitution of tyrosine 174 resulted in a total loss of invasive capacity, confirming the critical importance of this residue. Taken together these data suggest that PBF induces cell invasion and migration across a range of cell lines, and that this occurs via its interaction with cortactin.