Abstract P6-03-13: Inhibition of Src increases radioiodide uptake in breast cancer cells by inhibiting phosphorylation of pituitary tumor transforming gene binding factor (PBF)
Authors: Vikki L Poole, Martin L Read, Rachel J Watkins, Bhavika Modasia, Waraporn Imruetaicharoenchoke, Kristien Boelaert, Vicki E Smith, Christopher J McCabe
Journal: Cancer Research (2015)

Abstract Although not detectable in normal breast tissue, the sodium iodide symporter (NIS) has been found to be expressed in 70-80% of breast cancers. However, the majority of NIS is intracellular, leaving only 20-30% functional at the plasma membrane. Whilst radioiodine therapy has been proposed as a potential treatment for breast cancer, effective therapy would require increased levels of membranous NIS localisation in tumours. Previous work revealed that overexpression of pituitary tumor transforming gene binding factor (PBF) in thyroid cells leads to the redistribution of NIS from the plasma membrane into intracellular vesicles, thereby reducing radioiodide uptake, a process modulated by Src phosphorylation of PBF. Here we show that PBF and NIS have a consistent relationship in breast cancer, with phosphorylation of PBF at residue Y174 being critical for the association. Immunofluorescent microscopy revealed co-localisation between NIS and PBF in co-transfected MDA-MB-231, MCF-7 and T47D cells, with increased intracellular staining for NIS compared to cells transfected with NIS alone. Phosphorylated PBF was also observed to co-localise with NIS in T47D cells. Treatment with PP1, a Src inhibitor which modulates the phosphorylation of PBF, led to increased NIS plasma membrane staining and less intracellular co-localisation with PBF. Functional studies in MCF-7 and MDA-MB-231 cells demonstrated that PBF significantly repressed radioiodide uptake in cells expressing exogenous NIS (25% and 30% reduction respectively; n=3, p&amp;lt;0.05). Treatment with PP1 restored the ability of MCF-7 and MDA-MB-231 cells to uptake I-125 (1.24 and 1.69 fold increase respectively; n=3, p&amp;lt;0.05). Combined all-trans retinoic acid (ATRA) and dexamethasone treatment has previously been shown to enhance NIS expression and radioiodide uptake in MCF-7 cells. Importantly, in the face of NIS-induction via ATRA and dexamethasone, PBF retained its ability to repress iodide uptake (31% reduction; n=3, p&amp;lt;0.05). This repression was overcome by PP1 treatment, which restored radioiodide uptake to vector only transfected levels (1.22-fold increase; p=ns, n=3). In keeping with PP1 data, the Src inhibitors saracatinib and dasatinib inhibited the phosphorylation of PBF in MCF-7 and MDA-MB-231 cells. Radioiodide uptake studies revealed that 1nM dasatinib was capable of restoring the ability of MDA-MB-231 cells to uptake I-125 (2 fold increase; n=3, p&amp;lt;0.05) when expressing exogenous NIS and PBF compared to vehicle only treated cells. 10nM saracatinib was also capable of restoring I-125 uptake but with less potency than dasatinib. Inhibition of focal adhesion kinase (FAK) with PF573228 failed to ameliorate PBF’s repression on I-125 uptake, suggesting that FAK is not responsible for PBF phosphorylation. Taken together, these data suggest that PBF alters the subcellular location of NIS and potently represses its activity in breast cancer cells. Further, tyrosine phosphorylation of PBF by Src modulates the ability of breast carcinoma cell-lines to take up radioiodide, with important implications for adapting NIS as a potential therapy in breast cancer. Citation Format: Vikki L Poole, Martin L Read, Rachel J Watkins, Bhavika Modasia, Waraporn Imruetaicharoenchoke, Kristien Boelaert, Vicki E Smith, Christopher J McCabe. Inhibition of Src increases radioiodide uptake in breast cancer cells by inhibiting phosphorylation of pituitary tumor transforming gene binding factor (PBF) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-13.