Background Exploitation of the sodium iodide symporter (NIS) has potentially broad clinical application across different tumour ablative settings but often fails in aggressive cancer due to diminished transport activity. We aimed to discover whether enhancing NIS function by modulating proteostasis was targetable in vivo, as well as the clinical relevance to radioiodide (RAI) treatment of patients with cancer. Methods We used 3D modelling, iterative design, reformulation, RAI uptake, RNA-Seq, cell surface biotinylation assays and NanoBRET in transformed cell lines and primary thyroid cells from patients to identify new drugs targeted at enhancing NIS function and to uncover their respective mechanisms. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and SPECT/CT imaging in wild-type BALB/c and Tg-rtTA/tetO-BRAFV600E mice, as well as orthotopic NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) breast cancer. Findings Copper diethyldithiocarbamate (Cu(DDC)2), a metabolite of the FDA-approved drug disulfiram, modulated NIS function in thyroid and breast cancer cells (P < 0.05). Mechanistically, Cu(DDC)2 elicited a dual effect on NIS function, targeting valosin containing protein (VCP)—a key regulator of proteostasis—as well as inducing potent transcriptional responses (P < 0.05). In mice, the copper-bound metabolite stimulated NIS activity in normal thyroid tissue, thyroid tumours and in breast orthotopic tumours (P < 0.05), the latter augmented by the histone deacetylase inhibitor vorinostat (SAHA). Notably, there was clinical association of drug-perturbed genes in RAI-treated thyroid cancer, enabling construction of a robust dual risk score classifier for predicting recurrence (AUC >0.95; P < 0.001). Interpretation Our findings reveal a mechanistic pathway towards enhancing radionuclide uptake in vivo, with clinical relevance for RAI therapy and identifying survival indicators of recurrent disease. Funding This work was funded by the U.S. Department of Defense (BC201532P1), Medical Research Council (CiC/1001505 and MR/Z504828/1), British Thyroid Foundation (1002175). We further acknowledge support from the Wellcome Trust and EPSRC funded Centre for Medical Engineering at King’s College London (203148/Z/16/Z), the Wellcome Multiuser Equipment Radioanalytical Facility (212885/Z/18/Z), and the EPSRC programme for Next Generation Molecular Imaging and Therapy with Radionuclides (EP/S019901/1).

List of primers used for quantitative PCR.

Full list of significantly upregulated terms (q-value<0.05) in BrafV600E+K5-Tert tumors, compared to BrafV600E, from 20-week animals, employing the GSEA (gene set enrichment analysis) hallmarks of cancer database on RNAseq data.

Differentially expressed genes from RNAseq data when comparing BrafV600E vs. BrafV600E+K5-Tert tumors from 20-week animals.

Additional features of Braf and Braf+Tert murine tumors.

Additional insights from RNAscope analysis and telomere length evaluation. A-L. Analysis of intratumoral heterogeneity of Tert transcription via RNAscope.

Differentially expressed genes from RNAseq data

Full list of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis “up terms” for the BrafV600E vs. BrafV600E+K5-Tert comparison.

In vivo dabrafenib plus trametinib treatment of mice.

Sodium iodide symporter (NIS) expression in breast cancer renders radioiodide (RAI) a promising treatment modality. However, insufficient functional NIS within the plasma membrane limits RAI uptake (RAIU). We aimed to elucidate NIS regulatory mechanisms that impede RAIU in breast cancer and identify molecular targets for stimulating RAI-avidity in breast tumours. Mechanistic interaction between pituitary tumor-transforming gene-binding factor (PBF/PTTG1IP) and NIS was investigated through NanoBiT, co-immunoprecipitation, immunofluorescent microscopy, subcellular localisation and RAIU assays utilising wild-type and CRISPR-Cas9 PBF knockout breast cancer cells. In breast cancer cells, NIS:PBF interaction resulted in diminished RAIU, reversible through reduced PBF phosphorylation by the Src inhibitor dasatinib. Src overexpression diminished RAIU in a PBF-dependent manner that was mediated by Src myristoylation by N-myristoyltransferase 1 (NMT1). NMT1 inhibition significantly enhanced RAIU via Src and PBF in breast and thyroid cancer cells. Bioinformatic analyses revealed clinical associations between high Src and NMT1 expression and increased tumour recurrence in RAI-treated thyroid cancers indicating RAI-resistance. In breast cancer, high PBF and Src expression was associated with the more aggressive tumours that are most likely to benefit from targeted RAI therapy. We describe a new NIS regulatory pathway in breast cancer cells via Src myristoylation and PBF phosphorylation and show that the same pathway exists in thyroid cells, the canonical setting for the exploitation of NIS function. These findings reveal that PBF interaction with NIS may be modulated by Src, which in turn is susceptible to NMT inhibition, and suggest that targeting NMT1 may represent an innovative approach for augmenting RAI-avidity in breast cancer.

Supplementary TableS 1-7 and Figure Legends 1-5

Pairwise alignment of human:mouse telomerase reverse transcriptase (TERT) gene sequences.

Additional analyses of RNA sequencing data from mouse tumors with telomerase upregulation.

Lack of co-trafficking for VCP-dsRED and NIS-GFP suggests that the site of functional interaction between VCP and NIS is distant to the plasma membrane in HeLa cells

The sodium iodide symporter NIS is endosomally trafficked in association with clathrin

Fletcher A et al Supplementary Information

Fletcher A et al Data Source File

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Significant co-localization and trafficking of ARF4-dsRED and NIS-GFP proteins in co-incident vesicles at the plasma membrane in HeLa cells