lithium-chloride and Thyroid-Neoplasms

The Wnt/β-catenin signaling pathway is involved in the normal development of thyroid gland, but its disregulation provokes the appearance of several types of cancers, including papillary thyroid carcinomas (PTC) which are the most common thyroid tumours. The follow-up of PTC patients is based on the monitoring of serum thyroglobulin levels which is regulated by the thyroid transcription factor 1 (TTF-1): a tissue-specific transcription factor essential for the differentiation of the thyroid. We investigated whether the Wnt/β-catenin pathway might regulate TTF-1 expression in a human PTC model and examined the molecular mechanisms underlying this regulation. Immunofluorescence analysis, real time RT-PCR and Western blot studies revealed that TTF-1 as well as the major Wnt pathway components are co-expressed in TPC-1 cells and human PTC tumours. Knocking-down the Wnt/β-catenin components by siRNAs inhibited both TTF-1 transcript and protein expression, while mimicking the activation of Wnt signaling by lithium chloride induced TTF-1 gene and protein expression. Functional promoter studies and ChIP analysis showed that the Wnt/β-catenin pathway exerts its effect by means of the binding of β-catenin to TCF/LEF transcription factors on the level of an active TCF/LEF response element at [-798, -792 bp] in TTF-1 promoter. In conclusion, we demonstrated that the Wnt/β-catenin pathway is a direct and forward driver of the TTF-1 expression. The localization of TCF-4 and TTF-1 in the same area of PTC tissues might be of clinical relevance, and justifies further examination of these factors in the papillary thyroid cancers follow-up.

Topics: beta Catenin; Blotting, Western; Carcinoma, Papillary; Cell Differentiation; Chromatin Immunoprecipitation; DNA-Binding Proteins; Enhancer Elements, Genetic; Humans; Immunoenzyme Techniques; Lithium Chloride; Luciferases; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TCF Transcription Factors; Thyroid Neoplasms; Transcription Factors; Tumor Cells, Cultured; Wnt Proteins; Wnt Signaling Pathway

While representing only 3% of thyroid malignancies, medullary thyroid cancer (MTC) accounts for 14% of thyroid cancer deaths. MTC has a high rate of recurrence and lacks effective treatments. The histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) activate the Notch1 signaling pathway, while lithium chloride inhibits the glycogen synthase kinase-3ss (GSK-3ss) pathway. These compounds have been shown to limit growth and suppress hormonal secretion; thus, targeting different signaling pathways may be an effective treatment.. MTC cells were treated with varying combinations of up to 20 mM lithium chloride with either 3 mM VPA or 20 muM SBHA for 48 h. Western analysis was used to measure the effects on Notch1, GSK-3ss, and neuroendocrine (NE) markers. Growth was assessed by a methylthiazolyldiphenyl-tetrazolium (MTT) bromide cellular proliferation assay. Western analysis was used to determine the mechanism of growth regulation.. Combination therapy increased active Notch1, inhibited the GSK-3ss pathway, and decreased NE markers. Additive inhibition of growth was observed with combination therapy. Lower-dose combination therapy achieved greater decreases on NE markers and growth than treatment with any of the drugs alone. Moreover, an increase in the cleavage of the apoptotic markers caspase-3 and PARP was observed.. Combination therapy with lithium chloride and HDAC inhibitors suppresses NE markers and decreases growth via apoptosis of MTC cells in vitro. With the possibility of increased efficacy and decreased toxicity, combination therapy may represent a new strategy to treat MTC.

Topics: Cell Division; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lithium Chloride; Receptor, Notch1; Thyroid Neoplasms; Tumor Cells, Cultured; Up-Regulation; Valproic Acid

Glycogen synthase kinase-3beta (GSK-3beta) is an important regulator of cell proliferation and survival. Conflicting observations have been reported regarding the regulation of GSK-3beta and extracellular signal-regulated kinase (ERK1/2) in cancer cells. In this study, we found that raf-1 activation in human medullary thyroid cancer cells, TT cells, resulted in phosphorylation of GSK-3beta. Inactivation of GSK-3beta in TT cells with well-known GSK-3beta inhibitors such as lithium chloride (LiCl) and SB216763 is associated with both growth suppression and a significant decrease in neuroendocrine markers such as human achaete-scute complex-like 1 and chromogranin A. Growth inhibition by GSK-3beta inactivation was found to be associated with cell cycle arrest due to an increase in the levels of cyclin-dependent kinase inhibitors such as p21, p27, and p15. Additionally, LiCl-treated TT xenograft mice had a significant reduction in tumor volume compared with those treated with control. For the first time, we show that GSK-3beta is a key downstream target of the raf-1 pathway in TT cells. Also, our results show that inactivation of GSK-3beta alone is sufficient to inhibit the growth of TT cells both in vitro and in vivo.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Carcinoma, Medullary; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromogranin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Lithium Chloride; Maleimides; Mice; Mice, Nude; NIH 3T3 Cells; Phosphorylation; Proto-Oncogene Proteins c-raf; Signal Transduction; Thyroid Neoplasms; Xenograft Model Antitumor Assays

Lithium, clinically used in the treatment of bipolar disorders, is well known to induce thyroid growth. However, the mechanism involved is only incompletely characterized. Although it is conventionally believed that thyroid proliferation depends on the thyroid-stimulating hormone (TSH)/cAMP/cAMP response element binding protein (CREB) pathway, recent data indicate that Wnt/beta-catenin signalling may be of critical importance. In other cell types lithium activates canonical Wnt signalling by GSK-3beta inhibition, which in turn stabilizes cytosolic free beta-catenin. Here we investigated the potential modulation of Wnt/beta-catenin signalling under lithium treatment in primary and neoplastic human thyrocytes.. Primary (S18) and neoplastic (NPA, FTC133) thyrocytes treated with and without LiCl were analysed using Western blotting, immunoprecipitation, reporter-gene assay, MTT proliferation assay and transfection studies.. LiCl dose-dependently inhibited GSK-3beta, stabilized free beta-catenin and inhibited beta-catenin degradation. Furthermore, LiCl altered the assembly of adherens junction by upregulating the E-cad-herin repressor, Snail, and downregulated E-cadherin expression. At a dose of 5 mM, LiCl significantly increased the proliferative potency of thyrocytes, which appeared to be mediated by beta-catenin, since nuclear beta-catenin stimulated T-cell factor/lymphoid enhancer factor (TCF/LEF)-mediated transcription and upregulated downstream targets like cyclin D1. To characterize the specificity of Wnt/beta-catenin-driven thyrocyte proliferation, we transfected primary thyrocytes and FTC133 cells with dominant negative TCF4 to block Wnt-dependent pathways or with dominant negative CREB to inhibit the TSH/cAMP cascade. In cells transfected with dominant negative CREB lithium-stimulated proliferation was unchanged whereas blocking Wnt/beta-catenin by dominant negative TCF4 reduced proliferation by approx. 50%.. Our data indicate that Wnt/beta-catenin signalling is of major importance in the control of lithium-dependent thyrocyte proliferation.

Topics: beta Catenin; Blotting, Western; Cadherins; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cyclin D1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Signal Transduction; Thyroid Gland; Thyroid Neoplasms; Transfection; Wnt Proteins