Dissecting endocytic mechanisms reveals new molecular targets to enhance sodium iodide symporter activity with clinical relevance to radioiodide therapy

Objective: The sodium/iodide symporter (NIS) frequently shows diminished targeting to the plasma membrane (PM) in differentiated thyroid cancer (DTC), resulting in suboptimal radioiodide treatment and poor prognosis. However, the mechanisms which govern the endocytosis of NIS away from the PM are ill-defined and may have therapeutic potential. We previously showed that the proto-oncogene PBF binds NIS and enhances its internalisation and reported significant dysregulation of vesicular transport genes in DTC. Here, we challenged the hypothesis that the heterotetramer Adaptor Protein 2 (AP2) – a key regulatory factor in endocytosis – represents a critical target to modulate NIS function.

Methods: NanoBiT assays were used to assess the stringency of protein interactions. AP2 subunits a1, l2, and r2 were ablated via siRNA. The Cancer Genome Atlas (TCGA) was appraised for vesicular transport genes. Technetium-99m pertechnetate ^(99mTc) uptake was used to evaluate NIS function in vivo.

Results Acidic dipeptides are known to bind AP2r2.We identified a putative acidic dipeptide within the NIS C-terminus; abrogation of this (E578A/E579A) significantly increased ¹²⁵I uptake and retention of NIS at the PM as determined by immunofluorescent microscopy. Exogenous AP2r2 was confirmed to bind NIS in NanoBiT assays with mutation of r2 (R15H) increasing NIS binding affinity. Importantly, ablation of AP2 subunits a1 and l2 significantly increased radioiodide uptake and NIS protein expression. NanoBiT assays showed that AP2a1 and l2 ablation also increased NIS:PBF binding,whereas r2did not. The drug chloroquine induced radioiodide uptake rapidly in vitro (8hr), independently of its canonical influence on autophagy, which was blocked by ablation of AP2 suggesting an impact on NIS endocytosis. In vivo, chloroquine treatment of Balb/c mice significantly enhanced thyroidal uptake of ^99mTc, in combination with the HDACi SAHA. Appraisal of TCGA showed that higher expression of AP2 subunits (e.g. a2, b1) was associated with a significant reduction in disease-free survival in radioiodide (¹³¹I) treated DTC (n = 111; BRAF-like).

Conclusion: We propose that NIS internalisation is modulated by the interaction of a C-terminal diacidic motif with AP2. Given our mouse data, NIS internalisation may now be druggable in vivo with translatable potential to improve radioiodide therapy for patients with worse outcomes.