Manipulating NIS endocytosis as a druggable target to enhance radioiodide uptake and prognostic indictor of thyroid cancer recurrence

Background: Diminished sodium iodide symporter (NIS) activity at the plasma membrane (PM) is frequently associated with suboptimal radioiodide (RAI) uptake and poor prognosis in differentiated thyroid cancer (DTC). Endocytosis is a key determinant of symporter activity, with our recent study demonstrating specific interaction of NIS with Adaptor Protein 2 (AP2) – a central player in endocytosis. Here, our objective was to determine whether NIS endocytosis can be exploited as a druggable target to enhance RAI uptake, as well as identify predictive markers of recurrence.

Methods: Chloroquine (CQ) was used as a candidate endocytosis inhibitor. NIS localisation was quantified via NanoBRET and cell surface biotinylation assays (CSBA). NIS function was monitored by RAI (125I) uptake assays in vitro and technetium-99m pertechnetate (99mTc) uptake in wild-type BALB/c mice.

Results: CQ rapidly increased 125I uptake in TPC1-NIS (2.54-fold; P<0.0001) and 8505C-NIS (1.93-fold; P<0.05) cells peaking after 8 hr. CSBA confirmed elevated levels of cell-surface NIS in CQ-treated cells, which was supported in live CQ-treated cells via KRAS-NanoBRET assays. To challenge this, we ablated the endocytic factor PICALM, known to recruit AP2/clathrin to the PM, which prevented CQ induction of RAI uptake. in vivo, CQ treatment of BALB/c mice enhanced thyroidal 99mTc-uptake in combination with HDACi SAHA (52.7%; P=0.0003), as well as increasing NIS (2.2-fold; P<0.0001), TSHR (1.9-fold; P=0.001) and PAX8 mRNA expression (1.6-fold; P=0.003). Appraisal of TCGA identified 102 significantly dysregulated endocytic genes in RAI-treated DTC. Importantly, recurrent DTC had greater endocytic gene dysregulation, and higher AP2 expression. A predictive risk model using 30 AP2-related genes showed a markedly worse prognosis in high-risk RAI-treated DTC (Hazard Ratio=57.27,95%CI 16.49-198.87; P<0.001; n =137).

Conclusions: Our findings suggest that extensive dysregulation of endocytic genes results in NIS mislocalisation away from the PM, thereby reducing RAI uptake. We identify CQ as an FDA-approved pharmaceutical agent targeting NIS endocytosis, with translatable potential to improve RAI therapy.