Identification of novel sodium iodide symporter (NIS) interactors which modulate radioiodine uptake

Patients termed to have radioiodine-refractory differentiated thyroid cancer (RR-DTC) cannot accumulate sufficient radioiodine for a therapeutic response due to sodium iodide symporter (NIS) dysregulation via diminished expression and/or altered plasma membrane (PM) localisation. Currently, the regulation of NIS localisation remains poorly defined and despite protein-protein interactions being well-described to modulate trafficking events, the NIS interactome is limited. Previously, using mass spectrometry, we identified two novel functional NIS interactors – ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP). ARF4 overexpression and VCP knockdown approximately doubled radioiodine uptake in the TPC1 thyroid cancer cell line, with similar findings observed in human primary thyrocytes. In thyroid cancer, ARF4 was determined to be downregulated, and VCP overexpressed, providing a putative explanation for repressed NIS function. Now, we have determined that ARF4 does not alter NIS expression, but enhances membranous NIS localisation. Real-time imaging using total internal reflection fluorescence microscopy highlighted trafficking of NIS and ARF4 in co-incident vesicles at the PM, suggesting that ARF4 promotes NIS trafficking to the PM. The site of ARF4 binding was identified to be a VAPK motif within the NIS C-terminus; abrogation of this site resulted in a NIS protein whose function remained unaltered compared to wildtype NIS, but which could not be enhanced by ARF4. In contrast to ARF4, VCP decreased NIS protein expression, which was suggestive of a role for VCP in NIS processing and degradation. A panel of pharmacological VCP inhibitors – Eeyarestatin-1, NMS-873 and the FDA-approved Astemizole, Clotrimazole and Ebastine – all overcame VCP inhibition of NIS function, implicating the endoplasmic reticulum-associated degradation pathway as critical to NIS processing. The co-application of SAHA with VCP inhibitor Eeyarestatin-1 additively enhanced radioiodine uptake in vitro. Collectively we identify a novel potential therapeutic strategy for RR-DTC, based on already FDA-approved drugs.