BIOCHEMICAL ANALYSIS OF RADIOIODINE UPTAKE ENHANCEMENT IN ENDOCRINE CANCER

Abstract

Radioiodine treatment of differentiated thyroid cancer (DTC) relies on cellular uptake of 131I via the sodium iodide symporter (NIS). Many tumours, however, demonstrate NIS dysregulation, which is associated with a poorer prognosis. Breast cancers frequently express NIS, and hence radioiodine treatment may be a potential treatment option if the function can be optimised. Overexpression of the pituitary tumor-transforming gene-binding factor (PBF) has been reported previously to be responsible for reduced activity of NIS in thyroid and breast cancer. PBF interaction with NIS decreases membranous NIS localisation which reduces its function. The C-terminal tyrosine residue 174 (Y174) of PBF, which is phosphorylated by the protein kinase Src, is required for NIS binding and therefore radioiodine uptake can be modulated by overexpression and suppression of Src.

This thesis demonstrated that consistent mechanisms are apparent in thyroid and breast cancer cells, with PBF binding to NIS and reducing its membranous expression. CRISPR-Cas9 gene editing was used to knock out PBF in thyroid and breast cancer cell lines to functionally investigate the mechanisms of interaction between NIS, PBF, and Src. In both parental thyroid and breast cancer cells, overexpressed Src significantly reduced radioiodine uptake. Conversely, radioiodine uptake in cell lines in which PBF was deleted was not changed by Src overexpression, proving that Src’s impact upon radioiodine uptake is dependent upon PBF. In an alternative approach to overcoming Src/PBF-mediated NIS repression, this thesis also demonstrated that an N-myristoyltransferase inhibitor (NMTi) targeting Src myristoylation caused an increase in radioiodine uptake in parental thyroid and breast cancer cells. In contrast, the NMTi had no effect on thyroid cancer cells with siRNA-mediated Src knockdown or on PBF KO cells, establishing that the NMTi induces radioiodine uptake via Src and again confirming that the ability of Src to repress NIS activity is PBF dependent.

Src phosphorylation of PBF Y174 also mediates PBF stimulation of thyroid and breast cancer cell motility. A recent phosphoproteomic screen identified two potential proteins downstream of PBF overexpression, which are both involved in cell motility and regulated by Src. To gain further insight into the importance of PBF phosphorylation, a potential relationship between PBF and the small GTPase signalling proteins FGD-1 and N-WASP was explored. This thesis demonstrates that PBF-induced cell migration and invasion are mediated by both FGD-1 and N-WASP.

Collectively, the data of this thesis suggest that in both thyroid and breast tumours Src-phosphorylated PBF contributes both to the lack of sensitivity to radioiodine uptake and to the induction of cellular invasion. Critically, the clinical use of Src inhibitors in thyroid tumours with high PBF expression may stimulate radioiodine uptake, and this alternative treatment approach might also be of utility for patients with breast tumours.