Characterization of EpCAM in thyroid cancer biology by three-dimensional spheroids in vitro model.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy. Nowadays, undifferentiated thyroid cancers (UTCs) are still lethal, mostly due to the insurgence of therapy resistance and disease relapse. These events are believed to be caused by a subpopulation of cancer cells with stem-like phenotype and specific tumor-initiating abilities, known as tumor-initiating cells (TICs). A comprehensive understanding of how to isolate and target these cells is necessary. Here we provide insights into the role that the protein Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker for other solid tumors, may have in TC biology, thus considering EpCAM a potential marker of thyroid TICs in UTCs. METHODS: The characterization of EpCAM was accomplished through Western Blot and Immunofluorescence on patient-derived tissue samples, adherent cell cultures, and 3D sphere cultures of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) cell lines. The frequency of tumor cells with putative tumor-initiating ability within the 3D cultures was assessed through extreme limiting dilution analysis (ELDA). EpCAM proteolytic cleavages were studied through treatments with different cleavages' inhibitors. To evaluate the involvement of EpCAM in inducing drug resistance, Vemurafenib (PLX-4032) treatments were assessed through MTT assay. RESULTS: Variable EpCAM expression pattern was observed in TC tissue samples, with increased cleavage in the more UTC. We demonstrated that EpCAM is subjected to an intense cleavage process in ATC-derived 3D tumor spheres and that the 3D model faithfully mimics what was observed in patient's samples. We also proved that the integrity of the protein appears to be crucial for the generation of 3D spheres, and its expression and cleavage in a 3D system could contribute to drug resistance in thyroid TICs. CONCLUSIONS: Our data provide novel information on the role of EpCAM expression and cleavage in the biology of thyroid TICs, and our 3D model reflects the variability of EpCAM cleavage observed in tissue samples. EpCAM evaluation could play a role in clinical decisions regarding patient therapy since its expression and cleavage may have a fundamental role in the switch to a drug-resistant phenotype of UTC cells.

Role of Epigenetic Therapy in the Modulation of Tumor Growth and Migration in Human Castration-Resistant Prostate Cancer Cells with Neuroendocrine Differentiation.

INTRODUCTION: Neuroendocrine transdifferentiation (NED) of prostate cancer (PC) cells is associated with the development of resistance to antiandrogen therapy and poor prognosis in patients with castration-resistant PC (CRPC). Many of the molecular events, involved in NED, appear to be mediated by epigenetic mechanisms. In this study, we evaluated the antitumor activity and epigenetic modulation of 2 epigenetic drugs, such as the demethylating agent 5-aza-2'-deoxycytidine (AZA) and the methyl donor S-adenosylmethionine (SAM), in 2 human CRPC cell lines with NED (DU-145 and PC-3). METHODS: The effects of AZA and SAM on cell viability, cell cycle, apoptosis, migration, and genome-wide DNA methylation profiling have been evaluated. RESULTS: Both drugs showed a prominent antitumor activity in DU-145 and PC-3 cells, through perturbation of cell cycle progression, induction of apoptosis, and inhibition of cell migration. AZA and SAM reversed NED in DU-145 and PC-3, respectively. Moreover, AZA treatment modified DNA methylation pattern in DU-145 cells, sustaining a pervasive hypomethylation of the genome, with a relevant effect on several pathways involved in the regulation of cell proliferation, apoptosis, and cell migration, in particular Wnt/ß-catenin. CONCLUSIONS: A relevant antitumor activity of these epigenetic drugs on CRPC cell lines with NED opens a new scenario in the therapy of this lethal variant of PC.

Extra-nuclear TERT counteracts oxidative stress and promotes progression in papillary thyroid carcinoma.

The reactivation of TERT is associated with poor outcome in papillary thyroid cancer (PTC). Extra-telomeric functions of TERT were reported, with a protective role against oxidative stress (OS). The aim of the present study was to explore the extra-nuclear TERT localization in PTC and its role in cancer progression. TERT nuclear export under OS were analyzed in K1 PTC cell line. We investigated the role of different TERT localizations using specific TERT constructs that limit its localization to the nucleus or to the mitochondria. The effect of SRC kinase inhibitor PP2, which reduces TERT nuclear export, was investigated as well. Moreover, TERT localization was analyzed in 39 PTC tissues and correlated with the genetic profile and the level of OS, DNA damage and apoptosis in the tumors and with the clinical characteristics of the patients. We demonstrated that TERT is exported from the nucleus in response to OS induced either from H2O2 or the BRAF inhibitor PLX4720. We proved that extra-nuclear TERT reduces mitochondrial OS and induces mitochondrial fragmentation. Moreover, limiting mitochondrial TERT localization reduced proliferation, migration, AKT phosphorylation and glycolysis and increased DNA damage and p21 expression. Finally, in PTC tissues the fraction of mitochondrial/nuclear TERT resulted inversely correlated with OS and p21 expression and associated with tumor persistence. In conclusion, our data indicate that extra-nuclear TERT is involved in reducing the effect of excessive OS, thus promoting cancer cell survival. Extra-nuclear TERT may thus represent a marker of cancer progression and a possible therapeutic target in PTC.

The length of FOXE1 polyalanine tract in congenital hypothyroidism: Evidence for a pathogenic role from familial, molecular and cohort studies.

Introduction: FOXE1 is required for thyroid function and its homozygous mutations cause a rare syndromic form of congenital hypothyroidism (CH). FOXE1 has a polymorphic polyalanine tract whose involvement in thyroid pathology is controversial. Starting from genetic studies in a CH family, we explored the functional role and involvement of FOXE1 variations in a large CH population. Methods: We applied NGS screening to a large CH family and a cohort of 1752 individuals and validated these results by in silico modeling and in vitro experiments. Results: A new heterozygous FOXE1 variant segregated with 14-Alanine tract homozygosity in 5 CH siblings with athyreosis. The p.L107V variant demonstrated to significantly reduce the FOXE1 transcriptional activity. The 14-Alanine-FOXE1 displayed altered subcellular localization and significantly impaired synergy with other transcription factors, when compared with the more common 16-Alanine-FOXE1. The CH group with thyroid dysgenesis was largely and significantly enriched with the 14-Alanine-FOXE1 homozygosity. Discussion: We provide new evidence that disentangle the pathophysiological role of FOXE1 polyalanine tract, thereby significantly broadening the perspective on the role of FOXE1 in the complex pathogenesis of CH. FOXE1 should be therefore added to the group of polyalanine disease-associated transcription factors.

Emerging Roles of DLK1 in the Stem Cell Niche and Cancer Stemness.

DLK1 is a maternally imprinted, paternally expressed gene coding for the transmembrane protein Delta-like homologue 1 (DLK1), a non-canonical NOTCH ligand with well-described roles during development, and tumor-supportive functions in several aggressive cancer forms. Here, we review the many functions of DLK1 as a regulator of stem cell pools and tissue differentiation in tissues such as brain, muscle, and liver. Furthermore, we review recent evidence supporting roles for DLK1 in the maintenance of aggressive stem cell characteristics of tumor cells, specifically focusing on central nervous system tumors, neuroblastoma, and hepatocellular carcinoma. We discuss NOTCH -dependent as well as NOTCH-independent functions of DLK1, and focus particularly on the complex pattern of DLK1 expression and cleavage that is finely regulated from a spatial and temporal perspective. Progress in recent years suggest differential functions of extracellular, soluble DLK1 as a paracrine stem cell niche-secreted factor, and has revealed a role for the intracellular domain of DLK1 in cell signaling and tumor stemness. A better understanding of DLK1 regulation and signaling may enable therapeutic targeting of cancer stemness by interfering with DLK1 release and/or intracellular signaling.

FAM83B is involved in thyroid cancer cell differentiation and migration.

FAM83B has been recently identified as an oncogene, but its role in thyroid cancers (TC) is still unclear. We examined the expression of FAM83B and its possible involvement in cell migration and differentiation, in neoplastic/normal thyroid tissues and in TC human cell lines. FAM83B expression in TC varies according to the tumor histotype, being significantly downregulated in more aggressive and metastatic tissues. FAM83B levels in cell lines recapitulate patients' samples variations, and its total and cytoplasmic levels decrease upon the induction of migration, together with an increase in its nuclear localization. Similar variations were detected in the primary tumor and in the metastatic tissues from a follicular TC. FAM83B knock down experiments confirmed its role in thyroid differentiation and cell migration, as demonstrated by the reduction of markers of thyroid differentiation and the increase of the mesenchymal marker vimentin. Moreover, the silencing of FAM83B significantly increased cells migration abilities, while not affecting the oncogenic RAS/MAPK/PI3K pathways. Our data indicate for the first time a role for FAM83B in TC cell differentiation and migration. Its expression is reduced in dedifferentiated tumors and its nuclear re-localization could favour distant migration, suggesting that FAM83B should be considered a possible diagnostic and prognostic biomarker.

Thyroid Cancer Stem-Like Cells: From Microenvironmental Niches to Therapeutic Strategies.

Thyroid cancer (TC) is the most common endocrine malignancy. Recent progress in thyroid cancer biology revealed a certain degree of intratumoral heterogeneity, highlighting the coexistence of cellular subpopulations with distinct proliferative capacities and differentiation abilities. Among those subpopulations, cancer stem-like cells (CSCs) are hypothesized to drive TC heterogeneity, contributing to its metastatic potential and therapy resistance. CSCs principally exist in tumor areas with specific microenvironmental conditions, the so-called stem cell niches. In particular, in thyroid cancer, CSCs' survival is enhanced in the hypoxic niche, the immune niche, and some areas with specific extracellular matrix composition. In this review, we summarize the current knowledge about thyroid CSCs, the tumoral niches that allow their survival, and the implications for TC therapy.

Thyrotropin Receptor p.N432D Retained Variant Is Degraded Through an Alternative Lysosomal/Autophagosomal Pathway and Can Be Functionally Rescued by Chemical Chaperones.

Background: Loss-of-function mutations of thyrotropin receptor (TSHR) are one of the main causes of congenital hypothyroidism. As for many disease-associated G-protein coupled receptors (GPCRs), these mutations often affect the correct trafficking and maturation of the receptor, thus impairing the expression on the cell surface. Several retained GPCR mutants are able to effectively bind their ligands and to transduce signals when they are forced to the cell surface by degradation inhibition or by treatment with chaperones. Despite the large number of well-characterized retained TSHR mutants, no attempts have been made for rescue. Further, little is known about TSHR degradation pathways. We hypothesize that, similar to other GPCRs, TSHR retained mutants may be at least partially functional if their maturation and membrane expression is facilitated by chaperones or degradation inhibitors. Methods: We performed in silico predictions of the functionality of known TSHR variants and compared the results with available in vitro data. Western blot, confocal microscopy, enzyme-linked immunosorbent assays, and dual luciferase assays were used to investigate the effects of degradation pathways inhibition and of chemical chaperone treatments on TSHR variants' maturation and functionality. Results: We found a high discordance rate between in silico predictions and in vitro data for retained TSHR variants, a fact indicative of a conserved potential to initiate signal transduction if these mutants were expressed on the cell surface. We show experimentally that some maturation defective TSHR mutants are able to effectively transduce Gs/cAMP signaling if their maturation and expression are enhanced by using chemical chaperones. Further, through the characterization of the intracellular retained p.N432D variant, we provide new insights on the TSHR degradation mechanism, as our results suggest that aggregation-prone mutant can be directed toward the autophagosomal pathway instead of the canonical proteasome system. Conclusions: Our study reveals alternative pathways for TSHR degradation. Retained TSHR variants can be functional when expressed on the cell surface membrane, thus opening the possibility of further studies on the pharmacological modulation of TSHR expression and functionality in patients in whom TSHR signaling is disrupted.

Hypoxia-induced release, nuclear translocation, and signaling activity of a DLK1 intracellular fragment in glioma.

Glioblastoma multiforme is characterized in part by severe hypoxia associated with tumor necrosis. The cellular response to hypoxia can influence several properties of tumor cells associated with aggressive tumor growth, including metabolic adaptations and tumor cell migration and invasion. Here, we found that Delta Like Non-Canonical Notch Ligand 1 (DLK1) expression was elevated as compared with normal brain in a genetically engineered mouse model of glioma, and that DLK1 expression increased with tumor grade in human glioma samples. DLK1 expression was highest in hypoxic and perivascular tumor areas, and we found that hypoxia induced the release and nuclear translocation of an intracellular fragment of DLK1 in murine glioma as well as in human glioma cultures. Release of the intracellular fragment was dependent on ADAM17 and Hypoxia-inducible Factor 1alpha and 2alpha (HIF-1alpha/HIF-2alpha), as ADAM17 inhibitors and HIF1A/HIF2A siRNA blocked DLK1 cleavage. Expression of a cleavable form of DLK1 amplified several hypoxia-induced traits of glioma cells such as colony formation, stem cell marker gene expression, a PI3K-pathway-mediated metabolic shift, and enhanced invasiveness. Effects of DLK1 were dependent on DLK1-cleavage by ADAM17, as expression of non-cleavable DLK1 could not replicate the DLK1-induced hypoxic phenotype. Finally, forced expression of DLK1 resulted in more invasive tumor growth in a PDGFB-induced glioma mouse model without affecting overall survival. Together, our findings suggest a previously undescribed role for DLK1 as an intracellular signaling molecule.

8-Cl-cAMP and PKA I-selective cAMP analogs effectively inhibit undifferentiated thyroid cancer cell growth.

The main purpose of our work was to evaluate the effects of different cyclic adenosine monophosphate analogs on thyroid cancer-derived cell lines. In particular we studied 8-chloroadenosine-3',5'-cyclic monophosphate, the most powerful cyclic adenosine monophosphate analog, and the protein kinase A I-selective combination of 8-hexylaminoadenosine-3',5'cyclic monophosphate and 8-piperidinoadenosine-3',5'-cyclic monophosphate. The cyclic adenosine monophosphate/protein kinase A pathway plays a fundamental role in the regulation of thyroid cells growth. Site-selective cyclic adenosine monophosphate analogs are a class of cyclic adenosine monophosphate-derivate molecules that has been synthesized to modulate protein kinase A activity. Although the cyclic adenosine monophosphate/protein kinase A pathway plays a fundamental role in the regulation of thyroid cells proliferation, there are currently no studies exploring the role of cyclic adenosine monophosphate analogs in thyroid cancer. We evaluated the effects on cell proliferation, apoptosis activation and alterations of different intracellular pathways using 3-(4,5-dimetylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytofluorimetry, western blotting, and kinase inhibitors. Our results show that both compounds have antiproliferative potential. Both treatments were able to modify protein kinase A RI/RII ratio, thus negatively influencing cancer cells growth. Moreover, the two treatments differentially modulated various signaling pathways that regulate cell proliferation and apoptosis. Both treatments demonstrated interesting characteristics that prompt further studies aiming to understand the intimate interaction between different intracellular pathways and possibly develop novel anticancer therapies for undifferentiated thyroid cancer.

Multicellular spheroids from normal and neoplastic thyroid tissues as a suitable model to test the effects of multikinase inhibitors.

Multicellular three-dimensional (3D) spheroids represent an experimental model that is intermediate in its complexity between monolayer cultures and patients' tumor. In the present study, we characterize multicellular spheroids from papillary (PTC) and follicular (FTC) thyroid cancers and from the corresponding normal tissues. We show that these 3D structures well recapitulate the features of the original tissues, in either the differentiated and "stem-like" components. As a second step, we were aimed to test the effects of a small multikinase inhibitor, SP600125 (SP), previously shown to efficiently induce cell death in undifferentiated thyroid cancer monolayer cultures. We demonstrate the potent effect of SP on cell growth and survival in our 3D multicellular cultures. SP exerts its main effects through direct and highly significant inhibition of the ROCK pathway, known to be involved in the regulation of cell migration and ß-catenin turnover. Consistently, SP treatment resulted in a significant decrease in ß-catenin levels with respect to basal conditions in tumor but not in normal spheroids, indicating that the effect is promisingly selective on tumor cells.In conclusion, we provide the morphological and molecular characterization of thyroid normal and tumor spheroids. In this 3D model we tested in vitro the effects of the multikinase inhibitor SP and further characterized its mechanism of action in both normal and tumor spheroids, thus making it an ideal candidate for developing new drugs against thyroid cancer.

The cAMP analogs have potent anti-proliferative effects on medullary thyroid cancer cell lines.

The oncogenic activation of the rearranged during transfection (RET) proto-oncogene has a main role in the pathogenesis of medullary thyroid cancer (MTC). Several lines of evidence suggest that RET function could be influenced by cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity. We evaluated the in vitro anti-tumor activity of 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) and PKA type I-selective cAMP analogs [equimolar combination of the 8-piperidinoadenosine-3',5'-cyclic monophosphate (8-PIP-cAMP) and 8-hexylaminoadenosine-3',5'-cyclic monophosphate (8-HA-cAMP) in MTC cell lines (TT and MZ-CRC-1)]. 8-Cl-cAMP and the PKA I-selective cAMP analogs showed a potent anti-proliferative effect in both cell lines. In detail, 8-Cl-cAMP blocked significantly the transition of TT cell population from G2/M to G0/G1 phase and from G0/G1 to S phase and of MZ-CRC-1 cells from G0/G1 to S phase. Moreover, 8-Cl-cAMP induced apoptosis in both cell lines, as demonstrated by FACS analysis for annexin V-FITC/propidium iodide, the activation of caspase-3 and PARP cleavage. On the other hand, the only effect induced by PKA I-selective cAMP analogs was a delay in G0/G1-S and S-G2/M progression in TT and MZ-CRC-1 cells, respectively. In conclusion, these data demonstrate that cAMP analogs, particularly 8-Cl-cAMP, significantly suppress in vitro MTC proliferation and provide rationale for a potential clinical use of cAMP analogs in the treatment of advanced MTC.

SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways.

Thyroid cancer is the most common endocrine malignancy with increasing incidence worldwide.The majority of thyroid cancer cases are well differentiated with favorable outcome. However, undifferentiated thyroid cancers are one of the most lethal human malignancies because of their invasiveness, metastatization and refractoriness even to the most recently developed therapies.In this study we show for the first time a significant hyperactivation of ROCK/HDAC6 pathway in thyroid cancer tissues, and its negative correlation with p53 DNA binding ability.We demonstrate that a small compound, SP600125 (SP), is able to induce cell death selectively in undifferentiated thyroid cancer cell lines by specifically acting on the pathogenic pathways of cancer development. In detail, SP acts on the ROCK/HDAC6 pathway involved in dedifferentiation and invasiveness of undifferentiated human cancers, by restoring its physiological activity level. As main consequence, cancer cell migration is inhibited and, at the same time, cell death is induced through the mitotic catastrophe. Moreover, SP exerts a preferential action on the mutant p53 by increasing its DNA binding ability. In TP53-mutant cells that survive mitotic catastrophe this process results in p21 induction and eventually lead to premature senescence. In conclusion, SP has been proved to be able to simultaneously block cell replication and migration, the two main processes involved in cancer development and dissemination, making it an ideal candidate for developing new drugs against anaplastic thyroid cancer.

Loss-of-Function Variants in a Hungarian Cohort Reveal Structural Insights on TSH Receptor Maturation and Signaling.

CONTEXT: Congenital hypothyroidism (CH) is one of the most common inborn endocrine disorders with genetic background. Despite the well-established newborn CH screening program in Hungary, no systematic examination of the underlying genetic alterations has been performed as yet. OBJECTIVE: We aimed to explore TSH receptor (TSHR) mutations in a cohort of Hungarian patients with CH. PATIENTS: Eighty-five unrelated patients with permanent primary CH, all diagnosed at newborn screening, were selected. MAIN OUTCOME MEASURES: Coding exons of the TSHR gene were sequenced and evaluated together with the thyroid-specific clinical parameters. Functional features of the novel mutations were experimentally examined, and their comparative molecular models were built. RESULTS: In four patients (one heterozygous and three compound heterozygous), seven TSHR mutations were identified. Among these, N432(1.50)D and P449(2.39)L are novel missense alterations. Importantly, the N432(1.50) residue is highly conserved among G protein-coupled receptors, and its function has not been examined yet in human glycoprotein hormone receptors. Our results indicate that the N432(1.50)D mutation disrupts important, architecture-stabilizing intramolecular interactions and ultimately leads to the complete intracellular retention of the receptor. On the other hand, P449(2.39) is located in the intracellular part of the receptor, which is important in G protein coupling. The P449(2.39)L mutation results in signaling impairment, with a more profound effect on the Gq/11 pathway. CONCLUSION: TSHR mutations are common among Hungarian patients with CH. The novel genetic alterations revealed an important structural role of the N432(1.50) and the P449(2.39) residues in receptor expression and signaling, respectively.