Search

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.

Radioiodine treatment fails ≥25% of patients with thyroid cancer and has been proposed as a potential treatment for breast cancer. Cellular iodide uptake is governed by the sodium iodide symporter (NIS), which is frequently mislocalized in thyroid and breast tumours. However, the trafficking of NIS to the plasma membrane (PM) is ill-defined. Through mass spectrometry, co-immunoprecipitation, cell surface biotinylation and proximity ligation assays we identify two proteins which control NIS subcellular trafficking: ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP). HiLo microscopy revealed ARF4 enhanced NIS trafficking in co-incident PM vesicles, governed by a C-terminal VXPX motif, whilst papillary thyroid cancers (PTC) demonstrate repressed ARF4 expression. In contrast, VCP, the central protein in ER-associated degradation, specifically bound NIS and decreased its PM localization. Five chemically distinct allosteric VCP inhibitors all overcame VCP-mediated repression of NIS function. In mice, two re-purposed FDA-approved VCP inhibitors significantly enhanced radioiodine uptake into thyrocytes, whilst human primary thyrocytes showed similar increases. Critically, PTC patients with high tumoural VCP expression who received radioiodine had strikingly worse disease-free survival. These studies now delineate the mechanisms of NIS trafficking, and for the first time open the therapeutic possibility of systemically enhancing radioiodine uptake in patients via FDA-approved drugs.One Sentence SummaryNovel NIS interactors ARF4 and VCP alter NIS trafficking in vitro, and FDA-approved VCP inhibitors can significantly enhance radioiodine uptake.

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.

The obesity epidemic is associated with increases in the incidence of several types of cancer, including colorectal cancer, and is associated with poor outcomes for patients. Adipose tissue is considered biologically active and represents a plausible link between cancer and obesity due to the many factors that it secretes. In the present study, human adipose tissue was cultured in vitro and predifferentiated adipocyte secretome [preadipocyte (PAS)] and differentiated adipocyte secretome (DAS) were collected. Quantification of interleukin-6 (IL-6), leptin and hepcidin in the DAS medium was compared to the PAS medium. Fold change levels of hepcidin, leptin and IL-6 in DAS (2.88±0.28, 12.34±0.95 and 31.29±1.89 fold increases) were significantly higher compared to these in PAS (p=0.05). The SW480 colorectal cancer cells were co-cultured with DAS in the presence or absence of leptin, IL-6 or hepcidin inhibitors and cellular viability and proliferation assays were performed. The culture of SW480 with DAS increased the cell proliferation and viability by 30 and 15% (p=0.02 and p=0.03) respectively, which was reversed in the presence of inhibitors. Challenging the SW480 cells with IL-6 or hepcidin significantly elevated colonocyte‑secreted leptin (p=0.05). Challenging the SW480 cells with leptin or hepcidin resulted in elevated levels of colonocyte-secreted IL-6 (p=0.05). Similarly, challenging cells with either IL-6 or leptin markedly elevated the level of secreted hepcidin (p=0.05) and this was associated with an induction in colonocyte iron levels in both cases. Collectively, these data revealed that adipocyte-secreted factors can ultimately modulate colonocyte iron levels and phenotype.