Genetic alterations in the RTKs and PI3K/Akt and MAPK pathways are extremely prevalent in ATC and FTC, providing a strong genetic basis for an extensive role of these signaling pathways and the development of therapies targeting these pathways for ATC and FTC, particularly the former.

Tumor size is smaller in patients treated with metformin, suggesting inhibition of tumor growth by the drug. Among diabetics, the absence of metformin therapy is an independent factor for decreased likelihood of CR and increased risk of shorter progression-free survival. In vitro data suggest that p70S6K/pS6 is likely a molecular target of metformin in DTC cells.

Medullary thyroid cancer (MTC) is associated with activation of mammalian target of rapamycin (mTOR) signaling pathways. Recent studies showed that the antidiabetic agent metformin decreases proliferation of cancer cells through 5 0 -AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR. In the current study, we assessed the effect of metformin on MTC cells. For this purpose, we determined growth, viability, migration, and resistance to anoikis assays using two MTC-derived cell lines (TT and MZ-CRC-1). Expressions of molecular targets of metformin were examined in MTC cell lines and in 14 human MTC tissue samples. We found that metformin inhibited growth and decreased expression of cyclin D1 in MTC cells. Treatment with metformin was associated with inhibition of mTOR/p70S6K/pS6 signaling and downregulation of pERK in both TT and MZ-CRC-1 cells. Metformin had no significant effects on pAKT in the cell lines examined. Metformin-inducible AMPK activation was noted only in TT cells. Treatment with AMPK inhibitor (compound C) or AMPK silencing did not prevent growth inhibitory effects of metformin in TT cells. Metformin had no effect on MTC cell migration but reduced the ability of cells to form multicellular spheroids in nonadherent conditions. Immunostaining of human MTC showed over-expression of cyclin D1 in all tumors compared with corresponding normal tissue. Activation of mTOR/p70S6K was detected in 8/14 (57.1%) examined tumors. Together, these findings indicate that growth inhibitory effects in MTC cells are associated with downregulation of both mTOR/6SK and pERK signaling pathways. Expression of metformin's molecular targets in human MTC cells suggests its potential utility for the treatment of MTC in patients.

Metformin inhibits thyroid cancer cell growth. We sought to determine if variable glucose concentrations in medium alter the anti-cancer efficacy of metformin. Thyroid cancer cells (FTC133 and BCPAP) were cultured in high-glucose (20 mM) and low-glucose (5 mM) medium before treatment with metformin. Cell viability and apoptosis assays were performed. Expression of glycolytic genes was examined by real-time PCR, western blot, and immunostaining. Metformin inhibited cellular proliferation in high-glucose medium and induced cell death in low-glucose medium. In low-, but not in high-glucose medium, metformin induced endoplasmic reticulum stress, autophagy, and oncosis. At micromolar concentrations, metformin induced phosphorylation of AMP-activated protein kinase and blocked p-pS6 in low-glucose medium. Metformin increased the rate of glucose consumption from the medium and prompted medium acidification. Medium supplementation with glucose reversed metformin-inducible morphological changes. Treatment with an inhibitor of glycolysis (2-deoxy-D-glucose (2-DG)) increased thyroid cancer cell sensitivity to metformin. The combination of 2-DG with metformin led to cell death. Thyroid cancer cell lines were characterized by over-expression of glycolytic genes, and metformin decreased the protein level of pyruvate kinase muscle 2 (PKM2). PKM2 expression was detected in recurrent thyroid cancer tissue samples.In conclusion, we have demonstrated that the glucose concentration in the cellular milieu is a factor modulating metformin's anti-cancer activity. These data suggest that the combination of metformin with inhibitors of glycolysis could represent a new strategy for the treatment of thyroid cancer.

Tyrosine Kinase Inhibitors (TKIs) have been studied extensively in TC: sorafenib and lenvatinib have been approved by the FDA for the treatment of metastatic, RAI-refractory DTC, while vandetanib and cabozantinib are FDA approved for use in advanced MTC. Moreover, several additional TKIs, multi-targeted or specific, are currently under investigation in TC. The current manuscript provides an extensive review of the literature regarding targeted therapies in TC including the rationale behind their use, the clinical trials and an expert opinion on their use. Literature in English appearing at PubMed was thoroughly reviewed, especially manuscripts of the last 5 years. Expert commentary: Patients with advanced, progressive, metastatic TC should be evaluated for enrollment in a clinical trial or should be placed on treatment with one of the FDA- and EMA- approved agents.

NFV has a wide spectrum of activity against MTC cells, and its cytotoxicity can be augmented by inhibiting autophagy. Expression of NFV molecular targets in metastatic MTC suggests that NFV has a potential to become a thyroid cancer therapeutic agent.

To test the hypothesis that herpes viruses may have a role in thyroid neoplasia, we analysed thyroid tissues from patients with benign (44) and malignant (65) lesions for HSV1 and HSV2 DNA. Confirmatory studies included direct sequencing, analysis of viral gene expression, and activation of viral-inducible signalling pathways. Expression of viral entry receptor nectin-1 was examined in human samples and in cancer cell lines. In vitro experiments were performed to explore the molecular mechanisms underlying thyroid cancer cell susceptibility to HSV. HSV DNA was detected in 43/109 (39.4%) examined samples. HSV capsid protein expression correlated with HSV DNA status. HSV-positive tumours were characterized by activation of virus-inducible signalling such as interferon-beta expression and nuclear NFkappaB expression. Lymphocyte infiltration and oncocytic cellular features were common in HSV-positive tumours. HSV1 was detected with the same frequency in benign and malignant thyroid tumours. HSV2 was significantly associated with papillary thyroid cancer and the presence of lymph node metastases. The expression of HSV entry receptor nectin-1 was increased in thyroid tumours compared to normal thyroid tissue and further increased in papillary thyroid cancer. Nectin-1 expression was detected in all examined thyroid cancer cell lines. Nectin-1 expression in cancer cells correlated with their susceptibility to HSV. Inhibition of PI3K/AKT or MAPK/ERK signalling did not affect the level of nectin-1 expression but decreased thyroid cancer cell susceptibility to HSV. These findings showed that HSV is frequently detected in thyroid cancer. During tumour progression, thyroid cells acquire increased susceptibility to HSV due to increased expression of viral entry mediator nectin-1 and activation of mitogenic signalling in cancer cells.

The HIV protease inhibitor Nelfinavir (NFV) inhibits PI3K/AKT and MAPK/ERK signaling pathways, emerging targets in thyroid cancers. We examined the effects of NFV on cancer cells that derived from follicular (FTC), papillary (PTC) and anaplastic (ATC) thyroid cancers. NFV (1-20 µM) was tested in FTC133, BCPAP and SW1736 cell lines. The effects of NFV on cell proliferation were determined using real-time microscopy and by flow cytometry. DNA damage, apoptotic cell death and expression of molecular markers of epithelial-mesenchymal transition (EMT) were determined by Western blot and real-time PCR. Real-time imaging demonstrated that NFV (10 µM) increased the time required for the cell passage through the phases of cell cycle and induced DNA fragmentation. Growth inhibitory effects of NFV were associated with the accumulation of cells in G0/G1 phase, downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). NFV also induced the expression of γH2AX and p53BP1 indicating DNA damage. Treatment with NFV (20 µM) resulted in caspase-3 cleavage in all examined cells. NFV (20 µM) decreased the levels of total and p-AKT in PTEN-deficient FTC133 cells. NFV had no significant effects on total ERK and p-ERK in BRAF-positive BCPAP and SW1736 cells. NFV had no effects on the expression of EMT markers (Twist, Vimentin, E- and N-Cadherin), but inhibited the migration and decreased the abilities of thyroid cancer cells to survive in non-adherent conditions. We conclude that NFV inhibits proliferation and induces DNA damage in thyroid cancer cell lines. Our data suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.