Cell and Molecular Biology of Thyroid Disorders

The thyroid is a hormone gland involved in metabolism, regulatory functions, growth, and development of the human organism [...].

This Special Issue covers combined in vitro and animal studies [26,27] and single in vitro studies [28][29][30][31] investigating mechanisms or drug-induced changes in different thyroid cancer types. Proteomics technology was used to detect differences between the hypothyroid and euthyroid state in human subjects [32]. Review [23] Rusinek et al. [15] reviewed the current advantages of preoperative molecular diagnostic tests and the histopathological examination of DTC. In addition, they discussed the DTC therapy, as well as new treatment modalities for radioiodine-refractory DTC. This review discusses data regarding ATC. Another focus was MTC. The authors reviewed clinical management based on histopathology and the ret proto-oncogene (RET) mutation genotype, follow-up, and the prognostic marker serum calcitonin, and finally the available targeted therapy in advanced MTC [15].
Milling et al. [21] reviewed tyrosine kinase-inhibitor (TKI) therapy in MTC, which is a rare malignancy with a poor prognosis. In non-operable cases or for patients with tumor progression and metastases, systemic treatment is necessary. MTC is often insensitive to conventional chemotherapy, but the use of TKI, such as pazopanib, cabozantinib, and vandetanib, has shown promising results with an increase in progression-free survival and prolonged lifetime [21], but TKI can cause various adverse events (AEs). One common AE of this treatment is hypertension, which can potentially influence the well-being of the treated patients. The authors discuss the available treatment strategies of drug-induced hypertension.
This Special Issue also covers original studies on patient and cell culture samples, providing novel mechanistic insights into TC pathogenesis and new aspects that may be important for the prognosis of TC or may impact diagnosis and clinical therapy. Despite low mortality, well-differentiated thyroid carcinomas (WDTC) frequently relapse [16]. B-Raf (rapidly accelerated fibrosarcoma) proto-oncogene (BRAF) and Telomerase Reverse Transcriptase (TERT) mutations are related to the prognosis in TC. The authors showed that PFKFB2 promoter methylation analysis has potential ability to better stratify WDTC patients according to recurrence risk, independently of BRAF and TERT mutations. They concluded that DNA methylation analysis of PFKFB2 promoter could be a potential tool for estimating the risk of recurrence in WDTC patients, that can be easily performed using a low-cost technique compatible with the clinical practice of bisulfite pyrosequencing.
Calabrese et al. [17] published in vitro data showing that miR-19a overexpression in FTC-133 cells induces a more de-differentiated and aggressive phenotype. miR-19a seems to be involved a poorer prognosis of thyroid cancer. The authors propose that this miRNA could represent a prognostic factor and a valid therapeutic target in highly malignant anaplastic tumors. Studies on human tissue samples have to be performed in the future to prove the association of miR-19a expression with the relevant clinicopathological factors and prognosis [17].
Rusinek et al. [18] reported, in a second research article, the coexistence of TERT promoter mutations and the BRAF V600E alteration and its impact on the morphology of papillary TC in Polish patient samples [18]. TERTp hotspot mutations were highly correlated with the presence of the BRAF V600E mutation [18]. Their coexistence was significantly associated with sex, advanced patient age and disease stage, lymph node metastases, increased tumor size, and tumor-capsule infiltration. Although correlations were identified, the possibility of TERTp mutations being key molecular modulators responsible for PTC aggressiveness requires further study [18].
Lee et al. [19] applied whole exome sequencing (WES) to identify a novel Hedgehog-interacting protein G516R mutation in tissue samples from three patients with locally advanced PTC. WES indicated intra-tumor heterogeneity in locally advanced thyroid cancers. This study first identified HHIP G516R (G1546A), which promotes tumor aggressiveness in TC cells. The authors used a new technique suitable for identifying new potential therapeutic targets for TC [19].
An ex vivo (patient tissue samples) and in vitro study investigating whether osteopontin-a (OPN; OPNa splice variant) overexpression is associated with matrix calcification in papillary TC was performed by Ferreira et al. [20]. OPN splice variants are often associated with tumor progression in PTC. PTC samples with psammoma bodies revealed an increased OPN expression level. OPNa overexpression promotes higher matrix calcification and collagen synthesis. In response to OPN knockdown, calcification was inhibited, in parallel with the downregulation of calcification markers. OPNa was the main contributor to matrix calcification in the tested samples, providing a better understanding of the biology and ethiopathogenesis of the calcification process in PTC [20].
Starenki et al. [31] showed an up-regulation of the mitochondrial heat shock protein 70 mortalin in FTC, PTC, and ATC tumor cells. Mortalin promotes survival and proliferation of the thyroid cancer cells. Application of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) revealed that it can suppress ATC and PTC expressing RET/PTC or B-RafV600E.
Greco et al. [24] provide an overview of the imaging techniques used to date for both diagnosis and theranostic purposes in TC mouse models. These were developed to understand the fundamental mechanisms involved in tumorigenesis and to discover possible new targets. To date, many different approaches are available for imaging thyroid cancer in mouse models [24].
An in vivo and in vitro study focused on the impact of heme oxygenase-1 (HO-1) inhibitors on growth in mice tumor xenograft models and thyroid cancer cells [26]. HO-1 is overexpressed in thyroid cancer and is associated with tumor aggressiveness. The HO-1 inhibitors demonstrated therapeutic potential for inducing cell cycle arrest and promoting growth suppression of thyroid cancer cells [26].
Zhong et al. [27] investigated the anti-cancer effect of thiazolidinedione (TZD) and PPARγ agonist troglitazone and the statin lovastatin in human anaplastic thyroid cancer cells and in a mouse xenograft model [27]. The inhibitory effect of troglitazone/lovastatin is partly caused by cell cycle arrest (G0/G1 phase) and a decrease in hyperphosphorylated retinoblastoma protein signaling. These results support the hypothesis that the combination of troglitazone and lovastatin is a promising approach for treating ATC. The concept of statin-TZD therapy can be combined with other anti-cancer drugs [27].
In vitro studies have investigated the impact of simvastatin on anaplastic thyroid cancer cells [28]. The authors demonstrated that simvastatin inhibits the proliferation of RhoA/Rac1 protein by deactivation and overexpression of p21cip and p27kip, and reduced migration of the ATC cells [28].
Another in vitro study examined spheroids or thyrospheres, containing cancer stem-like cells, from B-CPAP and TPC-1 cell lines derived from PTC of the BRAF-like expression profile class, or stem-like cells from Nthy-ori 3-1 normal thyreocyte-derived cell line [29]. The authors recorded a significant decrease in glycolytic pathway metabolites and variations in Krebs cycle metabolites in three-dimensional (3D) aggregates versus parental cells. The authors demonstrated the metabolic profile of PTC cancer stem-like cells and suggested that metabolic changes are new biomarkers and targets for PTC therapy.
These findings are comparable to earlier data from studies investigating follicular thyroid cancer cells exposed to a Random Positioning Machine (RPM) [33]. Removing gravity forces resulted in changed concentrations of various glycolytic enzymes in FTC-133 follicular thyroid cells. Western blot analysis and flow cytometry revealed, for example, a reduction in alpha-enolase in FTC-133 follicular thyroid cancer cells grown for three days under microgravity conditions compared to static 1 g -samples [33].
A comparative proteome analysis of FTC-133 thyroid cancer cells, growing as a monolayer under normal gravity or within 3D spheroids under simulated microgravity realized by an RPM, showed an up-regulation of 69 proteins detected in spheroids [22]. Using semantic and in silico analyses, the authors showed that a high percentage of the 69 selected proteins had modifiable N6 lysine residues. This study shows a novel method to facilitate planning work on possible posttranslational modifications (PTMs) of the proteins of cells, actually changing their type of growth, and offers explanations for earlier results regarding the protein-lysine 6-oxidase (LOX) gene. A tremendous down-regulation of the LOX gene was observed in FTC-133 cells during the Shenzhou-8/SimBox space mission [34,35]. The protein-lysine 6-oxidase catalyzes deamination of lysine residues [36].
Krüger et al. [23] reviewed the current knowledge about TC and microgravity research. Microgravity influences processes such as apoptosis, the cytoskeleton, adhesion, the extracellular matrix, and influences cell growth [23,37]. FTC cells exposed to microgravity conditions shifted toward a less-malignant phenotype. Results from space medicine are important for rethinking conventional cancer research and may help pinpoint the cellular changes causing cancer. This knowledge may help develop novel therapies that will enhance the quality of life for patients or potentially help develop new countermeasures [23].
A further in vitro study focused on the impact of the mitogen-activated protein kinase (MEK) inhibitor selumetinib in different thyroid carcinoma cell lines [30]. Selumetinib significantly reduced cell viability. The drug restored the sodium iodide symporter (NIS) by inhibition of its related targeting miRNAs. Future studies to clarify the mechanism activated by hsa-miR-146b-5p, hsa-miR-146b-3p, and hsa-let7f-5p to stabilize NIS should be performed. Restoration of the NIS might be a new option to treat advanced radioiodine refractory differentiated TC [30].
This Special Issue also covers benign thyroid disorders. One study investigated the differences in the plasma proteome of patients with hypothyroidism before and after thyroid hormone substitution therapy [32]. Proteomics technology was applied to compare the plasma proteome between the hypothyroid and the euthyroid states in patients [32]. Changes in the expression of several acute-phase response proteins were detected. A pathway analysis revealed interleukin-6 and tumor necrosis factorα (TNF-α) as central factors and as dysregulated in hypothyroidism.
The aspects of glycoprotein functioning in thyrocyte physiology and thyroid disorders were reviewed for this issue [25]. Changes in glycan structures result in the progression of thyroid cancer and autoimmunity. Tumorigenesis is accompanied by changes in sialylation and fucosylation, β1,6-branching of glycans, the content and structure of poly-LacNAc chains, as well as O-GlcNAcylation. In thyroid autoimmunity, the main processes affected are sialylation and fucosylation. Thyroid glycobiology helps us to understand the role of glucose in disorders [25].
Overall, the 18 important contributions published in this Special Issue demonstrate novel findings in the field of thyroid research. I thank all the authors who contributed to this Special Issue, and I remain hopeful that the application of new molecular biological technologies will help with benign and malignant thyroid disorders and that the increasing knowledge of diagnosis, prevention, and new treatment strategies for TC, as well as the search for new proteins that may serve as new drug targets, will help reduce the incidence and mortality of advanced metastatic TC.

Conflicts of Interest:
The author declares no conflict of interest.