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The sodium iodide symporter (NIS) is a classical iodide pump typically localized within the cell plasma membrane in thyroid cells, where NIS expression is believed to ensure success of mainstay radioiodide therapy in thyroid cancers. Although radioiodide uptake is generally reduced in thyroid cancer tissue, intracellular nonmembranous NIS has been reported to increase, suggesting that NIS serves a pump-independent function. Thyroid cancer is one of the major component cancers of Cowden syndrome, a subset of which is caused by germline mutations in PTEN. In this study, we explored the noncanonical tumorigenic role of NIS in thyroid cancer cells in relation to PTEN signaling. PTEN knockdown in thyroid cancer cell lines stabilized intracellular NIS protein by promoting an interaction with NIS-LARG (leukemia-associated RhoA guanine exchange factor). Increased protein levels of cytoplasmic NIS enhanced RhoA activation and resulted in a promigration tumorigenic phenotype. Inhibition of NIS glycosylation through activation of the PI3K/AKT/mTOR signaling pathway contributed to mislocalization of NIS in the cytoplasm, facilitating its nonpump tumorigenic function through an interaction with LARG, which predominantly localized in the cytoplasm. Moreover, PTEN or PI3K/AKT/mTOR signaling could affect DPAGT1, a glycosylating enzyme involved in the initial step of N-linked glycosylation, to inhibit glycosylation of NIS. In summary, our results elucidate a pump-independent, protumorigenic role for NIS in thyroid cancer via its cross-talk with PTEN signaling.Significance: A novel pump-independent protumorigenic role of nonmembranous NIS challenges the presumption that radioiodine treatment of thyroid cancer is ineffective when transmembrane NIS is not expressed. Cancer Res; 78(21); 6121–33. ©2018 AACR.

Thyroid cancer is common, occurring in 1% of the general population. The relative frequencies of two of the most common subtypes of thyroid carcinoma, follicular (FTC) and papillary (PTC), vary depending on the regional prevalence of iodine deficiency. Although PTC has been more extensively studied, the etiology of sporadic FTC is poorly understood. To further elucidate this, we conducted microarray expression comparison of FTC tumors and normal thyroid tissue. Three commonly down-regulated genes, caveolin-1, caveolin-2, and GDF10/BMP3b, were chosen for further study on the basis of their localization to two chromosomal regions, 7q31.1 and 10q11.1, that commonly show loss of heterozygosity in FTC. Two additional genes, glypican-3 and a novel chordin-like gene, were also analyzed in view of their involvement in bone morphogenetic protein signaling and possible interaction with GDF10. Each of these five genes was down-regulated inor=15 of 19 FTC tumors (79%) by semiquantitative reverse transcription-PCR. Caveolin-1 showed preferential down-regulation of its beta-isoform at both the mRNA and protein level, suggesting a distinct function for this isoform. Caveolin-1 is of particular functional interest because it has been shown to interact with PTEN, the tumor suppressor gene mutated in Cowden syndrome, an inherited multiple hamartoma syndrome that includes predisposition to FTC. Immunohistochemical analysis of 141 thyroid tumors of various histological types showed significantly fewer caveolin-1-positive tumors in FTCs, including insular type tumors, and Hurthle cell carcinomas in comparison with normal thyroid. PTC and anaplastic thyroid carcinomas did not show significant down-regulation, and thus, caveolin-1 may become a useful molecular marker to differentiate the various histologies of thyroid malignancies.

Background: Na+/I− Symporter (NIS) is a glycoprotein expressed on the basolateral membrane of thyroid follicular cells and it facilitates iodide uptake from circulating blood. In thyroid cancer, NIS-mediated RAIU serves as the basis for therapeutic ablation of post-thyroidectomy remnants. However, patients with advanced thyroid cancer do not benefit from radioiodine therapy due to reduced or absence of NIS expression/function. Thus, it is of clinical importance to investigate the underlying mechanisms of NIS modulation such that strategies to selectively upregulate NIS expression/ function can be devised. Approach: Clinical and experimental evidences suggest an inverse correlation between Epithelial-to-mesenchymal transition (EMT) and NIS-mediated RAIU in thyroid cancer. To investigate if there is a functional role for EMT in decreasing RAIU, we determined the expression levels of 83 EMT-associated genes in PCCl3 rat thyroid cell lines treated with DMSO (control), Akti1/2 (small molecule inhibitor of pAkt1/2 known to increase RAIU) or TGF-β1 (cytokine known to decrease RAIU) using SABiosciences RT2 profiler EMT PCR array and subjected the results to Ingenuity Pathway Analysis (IPA). To mimic clinical conditions, cells were subjected to ≈5-day Thyroid Stimulating Hormone (TSH) withdrawal followed by acute stimulation by TSH for 24h. Cells were then treated for 24h with reagents in the presence of TSH followed by RNA extraction or RAIU assay or protein isolation. Results: From IPA analysis, we predicted apigenin, a plant derived flavonoid could potentially increase radioactive iodide uptake. Indeed, in-vitro RAIU assays and western blots revealed that apigenin (20μM) further enhances Akti1/2 (10μM)-induced RAIU up to 2-fold by increasing iodide influx and not by increasing NIS protein levels or decreasing iodide efflux in thyroid cells. pAkt inhibition was permissive for apigenin to increase RAIU. Apigenin (20μM) also rescued TGF-β1 (10ng/ml)-reduced RAIU up to 85%. Apigenin is known to affect many kinases and the molecular mechanism of action of apigenin in increasing RAIU is currently under investigation using candidate approach. Conclusion: Apigenin is the only compound reported so far that consistently further increases Akti1/2-induced RAIU. Apigenin promises to reveal novel molecular mechanisms of NIS modulation in thyroid. Apigenin also serves as a potential candidate for dietary supplement in combination with other drugs for future efficacy studies related to radioiodide therapy of thyroid cancer. Citation Format: Aparna Lakshmanan, Sissy Meihua Jhiang. Apigenin further enhances Akti1/2-induced radioactive iodine uptake (RAIU) and rescues TGF-β1-reduced RAIU in PCCl3 rat thyroid cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 87. doi:10.1158/1538-7445.AM2013-87