Antitumor activity of Koningic acid in thyroid cancer by inhibiting cellular glycolysis

Purpose Koningic acid (KA), a sesquiterpene lactone, has been identified as an antimicrobial agent. Recent studies have revealed KA’s antitumor activities in colorectal cancer, leukemia, and lung cancer. However, its antitumor effect in thyroid cancer remains largely unknown. Methods The effects of KA on proliferation, colony formation, apoptosis in thyroid cancer cells were assessed by MTT assay and flow cytometry. After KA treatment, the glycolysis ability of thyroid cancer cells was detected by ECAR, and the glycolytic products and relative ATP levels were measured by ELISA. The underlying mechanisms of antineoplastic activity of KA in thyroid cancer were detected by Western blot. Finally, the antineoplastic activity in vivo was observed in Xenograft mouse models. Results KA inhibited the proliferation, colony formation, and increased cell apoptosis in thyroid cancer cell lines in a dose and time-dependent manner. We verified that the glycolysis ability, ATP production, and lactic acid level in thyroid cancer cells had experienced an extensive decrease after KA treatment. In addition, lactic acid, the metabolite of glycolysis, could weaken the effect of KA on its colony formation ability in C643 thyroid cancer cell line. Our data also showed that KA kills thyroid cancer cells by inhibiting the MAPK/ERK pathway and decreasing Bcl-2 level. By contrast with the control group, the growth of xenograft tumor was dramatically inhibited by KA without obvious drug side effects. Conclusion Our data demonstrate that KA kills thyroid cancer cell lines by inhibiting their glycolysis ability, the MAPK/ERK pathway and the Bcl-2 level and suggest that KA has potential clinical value in thyroid cancer therapy.

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

Inhibition of the sonic hedgehog pathway activates TGF-β-activated kinase (TAK1) to induce autophagy and suppress apoptosis in thyroid tumor cells

The sonic hedgehog (Shh) pathway is highly activated in a variety of malignancies and plays important roles in tumorigenesis, tumor growth, drug resistance, and metastasis. Our recent study showed that the inhibitors of the Shh pathway such as cyclopamine (CP), a Smothened (SMO) inhibitor, and GANT61, a Gli1 inhibitor, have modest inhibitory effects on thyroid tumor cell proliferation and tumor growth. The objective of this study was to determine whether autophagy was induced by inhibition of the Shh pathway and could negatively regulate GANT61-induced apoptosis. Here we report that inhibition of the Shh pathway by Gli1 siRNA or by cyclopamine and GANT61 induced autophagy in SW1736 and KAT-18 cells, two anaplastic thyroid cancer cell lines; whereas Gli1 overexpression suppressed autophagy. Mechanistic investigation revealed that inhibition of the Shh pathway activated TAK1 and its two downstream kinases, the c-Jun-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). GANT61-induced autophagy was blocked by TAK1 siRNA and the inhibitors of TAK1 (5Z-7-oxozeaenol, 5Z), JNK (SP600125), and AMPK (Compound C, CC). Inhibition of autophagy by chloroquine and 5Z and by TAK1 and Beclin-1 siRNA enhanced GANT61-induced apoptosis and its antiproliferative activity. Our study has shown that inhibition of the Shh pathway induces autophagy by activating TAK1, whereas autophagy in turn suppresses GANT61-induced apoptosis. We have uncovered a previously unrecognized role of TAK1 in Shh pathway inhibition-induced autophagy and apoptosis.

Macrophage-Tumor Crosstalk in the Pathogenesis of Follicular Thyroid Cancer

Thyroid cancer is the most common endocrine malignancy and one of the fastest growing cancers in the United States. Follicular thyroid carcinoma (FTC) represents the second most common form of thyroid cancer diagnosed in the US and is most often tied to mutations in the RAS protein family of the MAP kinase pathway. In addition to driver mutations, FTC is characterized by a unique tumor microenvironment (TME) composed of cellular and non-cellular components that impact tumorigenesis and disease progression. Preliminary data from our lab has shown that CD45+ immune cells account for approximately 68% of all cells found in whole tumors collected from mouse models of RAS-driven disease. Macrophages account for the largest portion of known immune cell populations. Further experiments have demonstrated that tumor cell lines isolated from our RAS-driven models secrete cytokines known to impact the recruitment and activation state of cells of the myeloid lineage, particularly macrophages. However, it’s unclear what type of functional characteristics are induced by these secreted factors and how the resulting macrophage phenotype affects disease progression. Here, we sought to determine how bidirectional communication between macrophages and thyroid cancer cell lines could contribute to the development of a protumorigenic microenvironment. First, we began by defining how RAS-driven thyroid cancer cell lines affected the functional phenotype of previously unstimulated macrophages. Through gene expression analysis encompassing several markers of macrophage activation states, we determined that tumor cell-secreted factors induced the expression of multiple genes associated with tumor associated macrophages (TAM). In particular, we observed consistent upregulation of IL-10 and TNF-alpha, factors that have been associated with worsening disease. These results were further validated through quantification of protein secretion. In addition, we determined the role of activated macrophages in the progression of thyroid cancer, and specifically the effect of macrophage-secreted factors on tumor cell proliferation. Through direct and indirect assays of proliferation, we determined that factors secreted by classically-activated M1 macrophages inhibited cell proliferation. Surprisingly, secretions from alternatively-activated M2 macrophages reduced in vitro cell growth in some cell lines. Further analysis demonstrated that reduced cell proliferation was not associated with cell death, but rather was a result of delayed progression through the cell cycle. These results help to further define the macrophage phenotype within our model of FTC and will identify potential therapeutic targets to reduce the activity of protumorigenic cell populations.

Loss of primary cilia promotes mitochondria-dependent apoptosis in thyroid cancer

The primary cilium is well-preserved in human differentiated thyroid cancers such as papillary and follicular carcinoma. Specific thyroid cancers such as Hürthle cell carcinoma, oncocytic variant of papillary thyroid carcinoma (PTC), and PTC with Hashimoto’s thyroiditis show reduced biogenesis of primary cilia; these cancers are often associated the abnormalities in mitochondrial function. Here, we examined the association between primary cilia and the mitochondria-dependent apoptosis pathway. Tg-Cre;Ift88flox/flox mice (in which thyroid follicles lacked primary cilia) showed irregularly dilated follicles and increased apoptosis of thyrocytes. Defective ciliogenesis caused by deleting the IFT88 and KIF3A genes from thyroid cancer cell lines increased VDAC1 oligomerization following VDAC1 overexpression, thereby facilitating upregulation of mitochondria-dependent apoptosis. Furthermore, VDAC1 localized with the basal bodies of primary cilia in thyroid cancer cells. These results demonstrate that loss-of-function of primary cilia results in apoptogenic stimuli, which are responsible for mitochondrial-dependent apoptotic cell death in differentiated thyroid cancers. Therefore, regulating primary ciliogenesis might be a therapeutic approach to targeting differentiated thyroid cancers.

Stromal Interaction Molecule 1 (STIM1) Knock-down Attenuates Migration and Proliferation and Enhances the Expression of Thyroid Specific Proteins in Human Follicular Thyroid Cancer Cells.

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remained elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared with primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced migration, and expression of promigratory sphingosine 1-phosphate (S1P) and vascular endothelial growth factor-2 (VEGFR2) receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared with normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor (TSHR), thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated migration and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

CircHIPK3 Promotes Thyroid Cancer Tumorigenesis and Invasion through the Mirna-338-3p/RAB23 Axis

Objective：Thyroid cancer is a common type of endocrine malignancy, and its incidence has been steadily increasing in many regions of the world. Numerous studies have found that the circRNAs in various cancer types are aberrantly expressed, which could be potential biological diagnostic markers and therapeutic targets. The purpose of this study was to investigate the role of circHIPK3 in the development and progression of thyroid cancer and its mechanism. Subject and Methods：qRT-PCR was used to detect the relative expression levels of circHIPK3 in thyroid cancer cell lines (K1, CAL-62, TPC1), human thyroid normal cells (Nthy-ori 3-1), 10 pairs of thyroid cancer tissues and corresponding adjacent normal tissues. CCK-8 and Transwell assays were used to detect the proliferation and metastasis ability of cells. The targeted relationships between circHIPK3-miR-338-3p and miR-338-3p-RAB23 were predicted by bioinformatics analysis and verified by dual-luciferase reporter assays. Results and Conclusion: The downregulation of circHIPK3 significantly reduced the migration, invasion and proliferation of thyroid carcinoma. Then, we demonstrated that circHIPK3 up-regulated the expression of its target gene RAB23 by sponging miR-338-3p to promote the tumorigenesis and invasiveness of thyroid cancer. This study is the first to find that circHIPK3 plays the role of oncogenetic circRNA in thyroid cancer, which may provide new insights into how circRNA affects the progression of thyroid cancer. Our study also showed that circHIPK3 could be a novel biomarker for thyroid cancer.

Epithelial-to-mesenchymal transition and its association with PD-L1 and CD8 in thyroid cancer

Programmed cell death-ligand 1 (PD-L1) has recently been shown to play a role in the regulation of epithelial-to-mesenchymal transition (EMT); however, the relationship between PD-L1 expression, EMT and the inflammatory tumour microenvironment has yet to be investigated in thyroid cancer. To address this issue, we examined the expression of CD8, PD-L1 and the EMT markers E-cadherin and vimentin in a cohort of 74 papillary thyroid cancer (PTC) patients and investigated the association of these with clinicopathologic characteristics and disease-free survival (DFS). The relationship between PD-L1 and EMT was further examined in three thyroid cancer cell lines via Western blot and live cell imaging. In order to expand our in vitro findings, the normalised gene expression profiles of 516 thyroid cancer patients were retrieved and analysed from The Cancer Genome Atlas (TCGA). PD-L1 positivity was significantly higher in PTC patients exhibiting a mesenchymal phenotype (P = 0.012). Kaplan–Meier analysis revealed that PD-L1 (P = 0.045), CD8 (P = 0.038) and EMT status (P = 0.038) were all significant predictors for DFS. Sub-analysis confirmed that the poorest DFS was evident in PD-L1 positive patients with EMT features and negative CD8 expression (P < 0.0001). IFN-γ treatment induced upregulation of PD-L1 and significantly promoted an EMT phenotype in two thyroid cancer cell lines. Our findings suggest that PD-L1 signalling may play a role in stimulating EMT in thyroid cancer. EMT, CD8 and PD-L1 expression may serve as valuable predictive biomarkers in patients with PTC.