Towards enhancing sodium iodide symporter function in thyroid cancer: HiBiT tagging and CRISPR-driven drug discovery

Background: Sodium iodide symporter (NIS) is pivotal to iodide transport, and essential for therapeutic and imaging applications in differentiated thyroid cancer (DTC). However, its efficacy is often compromised by diminished activity at the plasma membrane (PM). We aimed to design a new high-throughput platform for screening drugs capable of enhancing endogenous NIS activity. Thus, we inserted an 11 amino acid HiBiT luminescence tag into 6 different locations within three extracellular loops of NIS and appraised their impact on radioiodine uptake.<br/><br/>Methods: HiBiT tag was inserted within the 1st, 3rd and 6th extracellular loops of NIS, at positions adjacent to Leucine-85, Glycine-214, Threonine-221, Cysteine-483, Aspartic acid-513, and Arginine-516. All 6 constructs were transiently transfected and appraised via radioiodine uptake assays, Western blotting, and Nano-Glo extracellular detection, to validate their impact on NIS expression and function, in the presence and absence of drugs known to modulate NIS function.<br/><br/>Results: Three plasmids retained functional iodide uptake in both TPC1 and Hela cells. Specifically, positions Cysteine-483, Aspartic acid-513, and Arginine-516 transported iodide, with increases of 2.8-fold, 3.9-fold, and 2.6-fold in TPC1 cells, and 6.1-fold, 6.9-fold, and 6.6-fold in Hela cells, respectively, compared to the pcDNA 3.1 vector (P&lt;0.05 in TPC1; P&lt;0.01 in Hela). Luminescence assays reinforced these findings, confirming that HiBiT-tagged NIS correctly localised to PM. Drug treatments – including the HDACi SAHA – induced parallel increases in iodide uptake and luminescence, confirming functionality of HiBiT-tagged NIS.<br/><br/>Implications: These proof-of-principle data suggest that HiBiT-tagging of NIS, particularly within the 6th extracellular loop, represents a viable strategy for progressing to CRISPR-mediated gene editing to facilitate the development of cell lines with endogenously tagged NIS, and hence novel high-throughput drug screening. This would pave the way for the first HTS of endogenous NIS activity, with the ultimate potential to improve therapeutic strategies for patients with refractory thyroid cancer.