Combined Vorinostat and Chloroquine Inhibit Sodium-Iodide Symporter Endocytosis and Enhance Radionuclide Uptake In Vivo.

PURPOSE: Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo. EXPERIMENTAL DESIGN: Informed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake, and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice. RESULTS: We identified an acidic dipeptide within the NIS C-terminus that mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine (CQ) modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/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. CONCLUSIONS: NIS internalization is specifically druggable in vivo. Our data, therefore, provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. See related commentary by Lechner and Brent, p. 1220.

Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter.

The sodium iodide symporter (NIS) functions to transport iodide and is critical for successful radioiodide ablation of cancer cells. Approaches to bolster NIS function and diminish recurrence post-radioiodide therapy are impeded by oncogenic pathways that suppress NIS, as well as the inherent complexity of NIS regulation. Here, we utilize NIS in high-throughput drug screening and undertake rigorous evaluation of lead compounds to identify and target key processes underpinning NIS function. We find that multiple proteostasis pathways, including proteasomal degradation and autophagy, are central to the cellular processing of NIS. Utilizing inhibitors targeting distinct molecular processes, we pinpoint combinatorial drug strategies giving robust >5-fold increases in radioiodide uptake. We also reveal significant dysregulation of core proteostasis genes in human tumors, identifying a 13-gene risk score classifier as an independent predictor of recurrence in radioiodide-treated patients. We thus propose and discuss a model for targetable steps of intracellular processing of NIS function.

Recurrence of Papillary Thyroid Cancer: A Systematic Appraisal of Risk Factors.

CONTEXT: Thyroid cancer recurrence is associated with increased mortality and adverse outcomes. Recurrence risk is currently predicted using clinical tools, often restaging patients after treatment. Detailed understanding of recurrence risk at disease onset could lead to personalized and improved patient care. OBJECTIVE: We aimed to perform a comprehensive bioinformatic and experimental analysis of 3 levels of genetic change (mRNA, microRNA, and somatic mutation) apparent in recurrent tumors and construct a new combinatorial prognostic risk model. METHODS: We analyzed The Cancer Genome Atlas data (TCGA) to identify differentially expressed genes (mRNA/microRNA) in 46 recurrent vs 455 nonrecurrent thyroid tumors. Two exonic mutational pipelines were used to identify somatic mutations. Functional gene analysis was performed in cell-based assays in multiple thyroid cell lines. The prognostic value of genes was evaluated with TCGA datasets. RESULTS: We identified 128 new potential biomarkers associated with recurrence, including 40 mRNAs, 39 miRNAs, and 59 genetic variants. Among differentially expressed genes, modulation of FN1, ITGα3, and MET had a significant impact on thyroid cancer cell migration. Similarly, ablation of miR-486 and miR-1179 significantly increased migration of TPC-1 and SW1736 cells. We further utilized genes with a validated functional role and identified a 5-gene risk score classifier as an independent predictor of thyroid cancer recurrence. CONCLUSION: Our newly proposed risk model based on combinatorial mRNA and microRNA expression has potential clinical utility as a prognostic indicator of recurrence. These findings should facilitate earlier prediction of recurrence with implications for improving patient outcome by tailoring treatment to disease risk and increasing posttreatment surveillance.

The potential interaction between medical treatment and radioiodine treatment success: A systematic review.

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.

Targeting Novel Sodium Iodide Symporter Interactors ADP-Ribosylation Factor 4 and Valosin-Containing Protein Enhances Radioiodine Uptake.

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.

PTTG and PBF Functionally Interact with p53 and Predict Overall Survival in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and poses a significant health burden due to its rising incidence. Although the proto-oncogene pituitary tumor-transforming gene 1 (PTTG) predicts poor patient outcome, its mechanisms of action are incompletely understood. We show here that the protein PBF modulates PTTG function, is overexpressed in HNSCC tumors, and correlates with significantly reduced survival. Lentiviral shRNA attenuation of PTTG or PBF expression in HNSCC cells with either wild-type or mutant p53, and with and without HPV infection, led to dysregulated expression of p53 target genes involved in DNA repair and apoptosis. Mechanistically, PTTG and PBF affected each other's interaction with p53 and cooperated to reduce p53 protein stability in HNSCC cells independently of HPV. Depletion of either PTTG or PBF significantly repressed cellular migration and invasion and impaired colony formation in HNSCC cells, implicating both proto-oncogenes in basic mechanisms of tumorigenesis. Patients with HNSCC with high tumoral PBF and PTTG had the poorest overall survival, which reflects a marked impairment of p53-dependent signaling.Significance: These findings reveal a complex and novel interrelationship between the expression and function of PTTG, PBF, and p53 in human HNSCC that significantly influences patient outcome. Cancer Res; 78(20); 5863-76. ©2018 AACR.

Pro-invasive Effect of Proto-oncogene PBF Is Modulated by an Interaction with Cortactin.

CONTEXT: Metastatic disease is responsible for the majority of endocrine cancer deaths. New therapeutic targets are urgently needed to improve patient survival rates. OBJECTIVE: The proto-oncogene PTTG1-binding factor (PBF/PTTG1IP) is overexpressed in multiple endocrine cancers and circumstantially associated with tumor aggressiveness. This study aimed to understand the role of PBF in tumor cell invasion and identify possible routes to inhibit its action. Design, Setting, Patients, and Interventions: Thyroid, breast, and colorectal cells were transfected with PBF and cultured for in vitro analysis. PBF and cortactin (CTTN) expression was determined in differentiated thyroid cancer and The Cancer Genome Atlas RNA-seq data. PRIMARY OUTCOME MEASURE: Pro-invasive effects of PBF were evaluated by 2D Boyden chamber, 3D organotypic, and proximity ligation assays. RESULTS: Our study identified that PBF and CTTN physically interact and co-localize, and that this occurs at the cell periphery, particularly at the leading edge of migrating cancer cells. Critically, PBF induces potent cellular invasion and migration in thyroid and breast cancer cells, which is entirely abrogated in the absence of CTTN. Importantly, we found that CTTN is over-expressed in differentiated thyroid cancer, particularly in patients with regional lymph node metastasis, which significantly correlates with elevated PBF expression. Mutation of PBF (Y174A) or pharmacological intervention modulates the PBF: CTTN interaction and attenuates the invasive properties of cancer cells. CONCLUSION: Our results demonstrate a unique role for PBF in regulating CTTN function to promote endocrine cell invasion and migration, as well as identify a new targetable interaction to block tumor cell movement.