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The sodium/iodide symporter (NIS) frequently shows diminished plasma membrane (PM) targeting in differentiated thyroid cancer (DTC), resulting in suboptimal radioiodide (RAI) treatment and poor prognosis. The mechanisms which govern the endocytosis of NIS away from the PM are ill-defined. Here, we challenged the hypothesis that new mechanistic understanding of NIS endocytosis would facilitate prediction of patient outcomes and enable specific drug modulation of RAI uptake in vivo. Through mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake and NanoBRET, we identify an acidic dipeptide within the NIS C-terminus which mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, chloroquine treatment of BALB/c mice significantly enhanced thyroidal uptake of99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. We propose that NIS internalisation is orchestrated by the interaction of a C-terminal diacidic motif with AP2σ2, together with the proto-oncogene PBF acting via AP2μ2. Given that NIS internalisation was specifically druggable in vivo, our data provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer.SummaryWe delineate the role of endocytic genes in regulating NIS activity at the plasma membrane and highlight the potential for systemic targeting of endocytosis to enhance radioiodine effectiveness in radioiodine-refractory cancer cells.

The sodium iodide symporter (NIS) is required for iodide uptake, which facilitates thyroid hormone biosynthesis. NIS has been exploited for over 75 years in ablative radioiodine (RAI) treatment of thyroid cancer, where its ability to transport radioisotopes depends on its localization to the plasma membrane. The advent of NIS-based in vivo imaging and theranostic strategies in other malignancies and disease modalities has recently increased the clinical importance of NIS. However, NIS trafficking remains ill-defined. Here, we used tandem mass spectrometry followed by coimmunoprecipitation and proximity ligation assays to identify and validate two key nodes—ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP)—controlling NIS trafficking. Using cell-surface biotinylation assays and highly inclined and laminated optical sheet microscopy, we demonstrated that ARF4 enhanced NIS vesicular trafficking from the Golgi to the plasma membrane, whereas VCP—a principal component of endoplasmic reticulum (ER)–associated degradation—governed NIS proteolysis. Gene expression analysis indicated VCP expression was particularly induced in aggressive thyroid cancers and in patients who had poorer outcomes following RAI treatment. Two repurposed FDA-approved VCP inhibitors abrogated VCP-mediated repression of NIS function, resulting in significantly increased NIS at the cell-surface and markedly increased RAI uptake in mouse and human thyroid models. Collectively, these discoveries delineate NIS trafficking and highlight the new possibility of systemically enhancing RAI therapy in patients using FDA-approved drugs.Significance:These findings show that ARF4 and VCP are involved in NIS trafficking to the plasma membrane and highlight the possible therapeutic role of VCP inhibitors in enhancing radioiodine effectiveness in radioiodine-refractory thyroid cancer.

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

Fletcher A et al Supplementary Information

Fletcher A et al Data Source File

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Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake. Cancer Res; 71(19); 6153–64. ©2011 AACR.

Thyroid cancer is the most common primary endocrine malignancy in adults and its incidence is rapidly increasing. Long non-coding RNAs (lncRNAs), generally defined as RNA molecules longer than 200 nucleotides with no protein-encoding capacity, are highly tissue-specific molecules that serve important roles in gene regulation through a variety of different mechanisms, including acting as competing endogenous RNAs (ceRNAs) that ‘sponge’ microRNAs (miRNAs). In the present study, using an integrated approach through RNA-sequencing of paired thyroid tumor and non-tumor samples, we have identified an interactome network between lncRNAs and miRNAs and examined the functional consequences in vitro and in vivo of one of such interactions. We have identified a likely operative post-transcriptional regulatory network in which the downregulated lncRNA, SPTY2D1-AS1, is predicted to target the most abundant and upregulated miRNAs in thyroid cancer, particularly miR-221, a well-known oncomiRNA in cancer. Indeed, SPTY2D1-AS1 functions as a potent tumor suppressor in vitro and in vivo, it is downregulated in the most advanced stages of human thyroid cancer, and it seems to block the processing of the primary form of miR-221. Overall, our results link SPTY2D1-AS1 to thyroid cancer progression and highlight the potential use of this lncRNA as a therapeutic target of thyroid cancer., This work was supported by grants SAF2016-75531-R and PID2019-105303RB-I00/AEI/10.13039/501100011033 from Ministerio de Ciencia e Innovación (MICIN); Fondo Europeo de Desarrollo Regional, B2017/BMD-3724 from Comunidad de Madrid; GCB14142311CRES from Asociación Española contra el Cáncer (AECC); and PI14/01980 from Instituto de Salud Carlos III (Spain). JR-M holds a FPU fellowship from MECD (Spain)

New drug approaches are urgently required to improve radioiodide (RAI) uptake for efficient ablation of thyroid cancer cells in RAI-refractory disease. Employing high-throughput screening of FDA-approved compounds we recently identified drugs capable of robust induction of sodium iodide symporter (NIS) activity to promote RAI uptake1. In particular, a leading drug candidate - the well-established anti-alcoholism drug disulfiram (DSF) - had not been previously implicated in regulating NIS. Here, we demonstrate that the ability of DSF to increase RAI-uptake in thyroid TPC-1 (3.1-fold; p 1Read ML, Brookes K, Thornton CEM, Fletcher A, Nieto HR, Alshahrani M, Khan R, Borges de Souza P, Zha L, Webster JRM, Alderwick LJ, Campbell MJ, Boelaert K, Smith VE, McCabe CJ (2022) Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter. Cell Chem Biol. 29(3):502-516.e7. Citation Format: Katie Brookes, Ling Zha, Jana Kim, Vinodh Kannappans, Weiguang Wang, Kavitha Sunassee, Philip J. Blower, Vicki E. Smith, Martin L. Read, Christopher J. McCabe. A critical role for copper in enhanced radioirdide uptake in thyroid cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5053.

Peer reviewed: True, INTRODUCTION: Radioactive iodine (RAI) therapy is a critical component in the post-surgical management of thyroid cancer patients, as well as being a central therapeutic option in the treatment of hyperthyroidism. Previous work suggests that antithyroid drugs hinder the efficacy of RAI therapy in patients. However, the effects of other background medications on RAI treatment efficacy have not been evaluated. Therefore, we performed a systematic review and meta-analysis investigating the potential off-target effects of medication on RAI therapy in patients with thyroid cancer and hyperthyroidism. METHODS: Systematic review and meta-analysis according to the 2020 PRISMA guidelines. Databases searched: MEDLINE, EMBASE and Cochrane Library for studies published between 2001 and 2021. RESULTS: Sixty-nine unique studies were identified. After screening, 17 studies with 3313 participants were included. One study investigated thyroid cancer, with the rest targeted to hyperthyroidism. The majority of studies evaluated the effects of antithyroid drugs; the other drugs studied included lithium, prednisone and glycididazole sodium. Antithyroid drugs were associated with negative impacts on post-RAI outcomes (n = 5 studies, RR = 0.81, p = 0.02). However, meta-analysis found moderate heterogeneity between studies (I2 = 51%, τ2 = 0.0199, p = 0.08). Interestingly, lithium (n = 3 studies), prednisone (n = 1 study) and glycididazole (n = 1 study) appeared to have positive impacts on post-RAI outcomes upon qualitative analysis. CONCLUSION: Our systematic review strengthens previous work on antithyroid medication effects on RAI, and highlights that this field remains under researched especially for background medications unrelated to thyroid disease, with very few papers on non-thyroid medications published. SYSTEMATIC REVIEW REGISTRATION: https://www.crd.york.ac.uk/prospero/display_record.php, identifier CRD42021274026.

New combinatorial drug strategies are urgently needed to improve radioiodine (RAI) uptake and efficiently ablate thyroid cancer cells, thereby reducing the risk of recurrent disease. Drug repurposing offers the promise of identifying already approved compounds capable of inducing sodium iodide symporter (NIS) function to enhance iodide uptake. However, a lack of thyroid cell-based assays amenable to high-throughput screening has limited progress. We utilised the mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide and screened the Prestwick Chemical Library (1200 drugs; 95% approved) for quenching of YFP fluorescence. This allowed us to identify putative candidate drugs which increased iodide uptake >2 SD above mean. Categorisation of these revealed a high proportion of drugs that modulate the proteostasis network (19/48; ~40%), including key processes in protein homeostasis such as endoplasmic reticulum-associated protein degradation (ERAD) and autophagy. Secondary screening validated the activity of proteostasis modulators in enhancing iodide uptake after ranking 73 leading compounds based on their pharmacologic (AUC, EMAX and EC50) and specificity of response (NIS+ve vs NIS-ve YFP-thyroid cells) at ten different drug doses (0.1 to 50 μM). Of importance, several repurposed drugs (e.g. ebastine, Prestwick N, Prestwick C and clotrimazole) in combination with the HDAC inhibitor vorinostat induced a robust enhancement in RAI uptake in thyroid cancer cells (TPC-1 and 8505C NIS+ve cells, up to 11-fold vs DMSO, P

BackgroundCell-free DNA (cfDNA) can be detected in the circulation of healthy individuals, but is found in higher concentrations in cancer patients. Furthermore, mutations in tumor cells can be identified in circulating DNA fragments. This has been the subject of significant interest in the field of cancer research, but little has been published in thyroid cancer.ObjectivesTo assess all available evidence on the use of circulating cfDNA in the diagnosis, management and surveillance of patients with differentiated thyroid cancer, and collate it into a systematic review to guide future research.MethodsA comprehensive literature search on the measurement of cfDNA in thyroid cancer was undertaken, and results from relevant studies collated into a systematic review.ResultsNine studies were identified, with varying methodologies and findings. Key techniques and findings are summarized.ConclusionThere is limited but promising evidence that somatic mutations in thyroid cancer can be detected in circulating cfDNA and are associated with more advanced disease. Further research is required to develop a clinically useful tool based on cfDNA to improve the management of thyroid cancers.

By exploiting the canonical function of the sodium iodide symporter (NIS), ablative radioiodine therapy is an effective treatment for papillary thyroid cancer (PTC). Unfortunately, ~25% of PTC patients are unable to accumulate therapeutically sufficient radioiodine due to diminished expression and/or altered plasma membrane localization of NIS. Patients with radioiodine-refractory PTC have reduced mean survival times. Radioiodine therapy has been proposed as a viable treatment for breast cancer but is hampered by low levels of membranous NIS localization. Currently, however, the regulation of NIS membrane localization remains ill-defined. Mass spectrometry identified the protein p97/VCP as a novel NIS interactor, which was validated through co-immunoprecipitation and proximity ligation assays. p97 siRNA depletion increased NIS-mediated radioiodine uptake by 97% and 141% in the lentivirally-expressing NIS MDA-MB-231 breast and TPC1 thyroid cancer cell lines respectively (p TCGA analyses of matched PTCs revealed p97 mRNA expression is highly upregulated in PTC compared to matched normal thyroid (n=58, p Our data therefore highlight a new pathway of NIS regulation. Critically, two of these p97 inhibitors are already FDA-approved, highlighting a novel potential therapeutic strategy for enhancing radioiodine uptake in patients with radioiodine-refractory PTC and increasing the feasibility of radioiodine therapy in breast cancer via the transient inhibition of p97 activity. Citation Format: Alice Fletcher, Martin L. Read, Vikki L. Poole, Vicki E. Smith, Christopher J. McCabe. p97/VCP: A novel interactor of the sodium iodide symporter, which can be pharmacologically targeted to increase radioiodine uptake in thyroid and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 920.

The PTTG1-binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione-S-transferase (GST) pull-down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126-132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53-responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (90%; P0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild-type TP53 and high PBF expression also had low p53 protein levels (P0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.

Most pituitary adenomas are slow-growing, benign tumours. Some do, however, demonstrate aggressive growth characteristics that include infiltration of the dura, bone, and surrounding tissues. Despite such apparent aggressive features, these invasive adenomas are not considered to be truly malignant. To make a diagnosis of pituitary carcinoma requires the presence of a pituitary tumour associated with central nervous system or systemic metastases. Little is known of the pathogenesis of these tumours and the prognostication of future tumour behaviour through clinical, histopathological, or molecular analyses remains challenging (1).

Healthcare payers are increasingly entering into innovative reimbursement agreements to manage the tension between funding new but expensive treatments, and obtaining value (measured in terms of clinical effectiveness, improved quality of care, health-related quality of life [HR-QOL], etc.) for money. These often commit substantial resources, and thus impose a significant opportunity cost on healthcare systems. Commentators and stakeholders are increasingly concerned that not all such approaches represent good value for money. However, there is little if any literature providing guidance on their design or evaluation. These reimbursement mechanisms have been given many names, such as ‘risk sharing’, ‘coverage with evidence development’, ‘field evaluations’ and ‘health impact guarantees’. We used the umbrella term ‘access with evidence development’ approaches (AEDs) to ensure discussions focussed upon the substantial issues common to all of these rather than their differences. The purpose of the Banff Summit (Alberta, Canada; 22–23 February 2009) was to bring together experts from around the world with direct experience of designing and implementing approaches to access with the aim of identifying principles to inform their design. The programme consisted of a presentation of findings from a review of the published literature and, drawing on that literature, a proposed checklist to describe and potentially evaluate such schemes. Participants from the US, the UK, Australia and Canadian provinces (British Columbia, Ontario and Alberta) gave presentations on their experience of AED approaches. Each talk was followed by an open discussion of the lessons that could be drawn from this experience. The final session considered whether it was possible to identify principles of good design in AED schemes. The Summit organizers were charged with producing a written statement of these principles for circulation to all participants. Each individual was then invited to sign up to the statement. We provide here the consensus statement from the summit. This is followed by the literature review completed in preparation for the meeting, a paper that outlines the descriptive framework (which was developed for the Summit) and an evaluation of an AED model implemented in the UK NHS using this framework. In addition, experts from the US and the UK write about the future development of AEDs in their respective healthcare systems and consider how adherence to the principles set out in the consensus statement would improve current and future AEDmodels.