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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.

Supplementary Figure S1. Loss of heterozygosity of Ch22q genes in poorly differentiated (PDTC) and anaplastic thyroid cancers (ATC). Supplementary Figure S2. Focal NF2-exon 4 homozygous deletion in KHM-5M anaplastic thyroid cancer cells. Supplementary Figure S3. FISH for NF2 in anaplastic thyroid cancers. Supplementary Figure S4. NF2/Merlin defects in thyroid cancer cell lines. Supplementary Figure S5. Merlin inhibits RAC1-PAK activity. Supplementary Figure S6. Merlin effects on EGFR signaling and growth of RAS mutant thyroid cancer cell lines. Supplementary Figure S7. PCR of genomic DNA from mouse thyroid tissues of the indicated genotypes with primers that distinguish wild-type from mutant Hras alleles. Supplementary Figure S8. TEAD1 is the most abundant TEAD isoform in thyroid cancer cells. Supplementary Figure S9. A) Left, Concentration-dependent growth inhibitory effects of the MEK inhibitor selumetinib (AZD6244) in RAS mutant thyroid cancer cell lines cells. Black, null/low NF2. Red, wt NF2. The IC50 of each cell line is shown in the boxed legend. Right, Western blots for merlin, pMEK and pERK in the cell lines studied. B) C643 cells expressing scrambled (pLKO.1) or shNF2 were treated with FRAX, AZD or their combination at the indicated concentrations (nM) in 1 percent of serum. Cells were counted at 6 days (*p

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

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

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

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.

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.

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

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.

The G1P1A[8] rotavirus vaccine candidate 89-12, the precursor to Rotarix, stimulated high titers of neutralizing antibodies to non-G1/P1A[8] serotypes of human rotavirus in naturally infected subjects before attenuation by cell-culture passages. These responses were greatly diminished in young infants (median age, 11 weeks) administered the attenuated vaccine. Because of the possibility of improved responses in older infants, the immunogenicity of the 89-12 vaccine candidate was evaluated after administration of 2 doses beginning at either 4 or 6 months of age. As was found in young infants, neutralizing antibody responses to non-G1/P1A[8] rotaviruses were considerably lower than those observed after natural infection. The reasons identified were overall (P

A quadrivalent precursor to the pentavalent rotavirus vaccine candidate RotaTeq was evaluated in a 3-dose, 439-subject study. To determine immunogenicity, the quantity of rotavirus immunoglobulin A (IgA) in stool specimens obtained, at 1 of 10 study sites, from 37 placebo and 37 vaccine recipients was measured. None of the placebo recipients showed a clinically important (>/=3-fold) increase in stool rotavirus IgA, whereas 31 vaccine recipients showed an increase after at least 1 dose of vaccine. In total, 16, 19, and 15 vaccine recipients had increases after 1, 2, and 3 doses, respectively, indicating that a 3-dose regimen increased the immune response elicited by this vaccine.

Summary Background Rotavirus is the most common cause of severe, dehydrating diarrhoea in infants worldwide. We assessed the safety, immunogenicity, and efficacy of a live, oral human rotavirus vaccine, 89–12, in US children in a randomised, placebo-controlled, double-blind multicentre trial. Methods 215 healthy infants were enrolled, of whom 213 were given two doses of 89–12 (containing 1×10 5 plaque-forming units) or placebo, and 213 were followed up through one rotavirus season. The frequency of side-effects was compared for 7 days after each dose of vaccine. Immune responses to rotavirus were assessed by serum and stool IgA, and by serum 89–12 neutralising titres. The primary outcome variable (protection from rotavirus disease) was evaluated by comparing the frequencies of rotavirus gastroenteritis in an intention-to-treat analysis. Findings Adverse reactions were mild. Low-grade fever (≥38·1°C) after the first dose was the only side-effect significantly more common in the vaccine group than in the placebo group (21 [19%] vs 5 [5%], p=0·001). An immune response to vaccine was detected in 94·4% of vaccinees. Rotavirus disease occurred in 18 of 107 placebo recipients and two of 108 vaccine recipients (vaccine efficacy 89·0% [95% Cl 65·4–94·5]). Ten infants in the placebo group but none in the vaccine group were presented for medical care. Interpretation The 89–12 rotavirus vaccine was safe and immunogenic and provided a high degree of protection against rotavirus disease. Further investigations of this vaccine are needed to confirm these findings in other settings.

The safety and immunogenicity of an orally administered live human rotavirus vaccine candidate (89-12), attenuated by 33 passages in monkey kidney cells, were evaluated in placebo-controlled trials in adults, children and infants. This strain was selected because natural infections with 89-12-like rotaviruses provided 100% protection over two years. The initial evaluations in adults, seropositive children and nine infants indicated that the vaccine was safe. Two doses of vaccine (10(5) p.f.u. dose-1) or placebo were then given to 42 infants, aged from 6 to 26 weeks. No significant difference in side effects was seen. Seroconversion was demonstrated in 19 of 20 previously uninfected vaccine recipients, but > or = 4-fold rises in 89-12 neutralizing antibody titers were detected in only seven subjects. Intestinal IgA responses were detected in 15 subjects. This attenuated human rotavirus was safe and immunogenic and should be further evaluated as a vaccine candidate.

The safety and immunogenicity of a booster dose of a new acellular pertussis component diphtheria-tetanus toxoids-pertussis vaccine (DTaP) were compared with whole cell pertussis component diphtheria-tetanus toxoids-pertussis vaccine (DTwP). Fifty children ages 15 to 18 months and 50 children ages 4 to 6 years were studied. The incidence of adverse reactions observed during the first 72 hours after vaccination in the DTaP/DTwP vaccinees were: pain, 32%/92% (P0.001); redness, 14%/24% (P = 0.2); swelling, 2%/14% (P0.03); fever, 52%/90% (P0.001); drowsiness, 14%/34% (P0.05); fussiness, 32%/88% (P0.001); and unusually poor appetite, 6%/42% (P0.001). The geometric mean titers of anti-pertussis toxin and anti-filamentous hemagglutinin antibody were also significantly (P0.001) higher in the DTaP compared to the DTwP recipients. When administered as a booster dose this DTaP vaccine, which has been chosen by the NIH for the second pertussis vaccine clinical efficacy trial, was more immunogenic for pertussis toxin and filamentous hemagglutinin and caused fewer and less severe adverse reactions compared with the Connaught DTwP vaccine used in this study.

To measure protection induced by natural rotavirus infection, 163 infants enrolled in a rotavirus vaccine trial were prospectively followed for 2 years. Serotype 1 rotaviruses were the predominant circulating strains during the study. Over the 2 years of observation, significantly fewer infants infected before enrollment developed a symptomatic reinfection (0 of 21) or any reinfection (4 of 21) compared with previously uninfected infants (P = .0003). Of the 60 infants who developed a primary rotavirus infection in the first year (40 symptomatic, 20 asymptomatic) only 4 were reinfected in the second year compared with 29 of 82 subjects not previously infected (P = .00003). Asymptomatic primary infection appeared to be as protective as symptomatic primary infection. The only symptomatic reinfections occurred in 2 subjects who did not develop rotavirus antibody after the initial detection of rotavirus. An age-related reduction in the ratio of symptomatic to asymptomatic primary rotavirus infection was also detected. In this study, protection against homotypic serotype 1 reinfection appeared to last greater than or equal to 2 years.