hes1-protein--human and Thyroid-Neoplasms

Dominant-activating mutations in the RET proto-oncogene, a receptor tyrosine kinase, are responsible for the development of medullary thyroid carcinoma (MTC) and causative for multiple endocrine neoplasia (MEN) type 2A and 2B. These tumors are highly aggressive with a high propensity for early metastasis and chemoresistance. This attribute makes this neoplasia an excellent model for probing mechanisms underlying cancer progression.. The expression level of miR-182 was measured in MTC tumor specimens and in TT cells by real-time RT-PCR. TT cells and modified NThy-ori 3.1 that stably express RETM918T were used to investigate RET-dependent regulation of miR-182. Identification and validation of miR-182 targets and pathways was accomplished with luciferase assays, qRT-PCR, Western blotting and immunofluorescence. In vitro, overexpression and knockdown experiments were carried out to examine the impact of miR-182 and HES1 on invasion and migration.. We found that miR-182 expression is significantly upregulated in MTC patient samples and tumor-derived cell lines harboring mutated RET. Inhibition of RET oncogenic signaling through a dominant-negative RET∆TK mutant in TT cells reduces miR-182, whereas overexpression of RETM918T in NThy-ori 3.1 cells increases miR-182 levels. We further show that overexpression of this miRNA in NThy.miR-182 cells promotes the invasive and migratory properties without affecting cell proliferation. MiR-182 is upregulated after RET induced NF-κB translocation into the nucleus via binding of NF-κB to the miR-182 promoter. Database analysis revealed that HES1, a repressor of the Notch pathway, is a target of miR-182, whose upregulation correlates with loss of HES1 transcription in MTC tissue samples and mutant RET cell lines. Moreover, we demonstrated that the 3'UTR of the HES1 mRNA bearing the targeting sequence for miR-182 clearly reduced luciferase reporter activity in cells expressing miR-182. Decreased expression of HES1 promotes migration by upregulating Notch1 inhibitor Deltex1 and consequent repression of Notch1.. We demonstrate a novel mechanism for MTC aggressiveness in which mutated RET/NF-κB-driven expression of miR-182 impedes HES1 activation in a negative feedback loop. This observation might open new possibilities to treat RET oncogene associated metastatic cancer.

Topics: 3' Untranslated Regions; Carcinoma, Neuroendocrine; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Models, Biological; Neoplasm Invasiveness; NF-kappa B; Proto-Oncogene Mas; Proto-Oncogene Proteins c-ret; Receptor, Notch1; RNA Interference; Signal Transduction; Thyroid Neoplasms; Transcription Factor HES-1

Anaplastic thyroid cancer (ATC) is characterized by very aggressive growth with undifferentiated features. Recently, it has been reported that the Notch1 signaling pathway, which affects thyrocyte proliferation and differentiation, is inactivated in ATC. However, it remains largely unknown whether using Notch1 activating compounds can be an effective therapeutic strategy in ATC. Therefore, in this study, we aimed to evaluate the drug effects of a potential Notch activator hesperetin on ATC cell.. A unique ATC cell line HTh7 was used to evaluate the drug effects of hesperetin. The Notch1 activating function and cell proliferation were evaluated. The mechanism of growth regulation was investigated by the detection of apoptotic markers. The expression levels of thyrocyte-specific genes were quantified for ATC redifferentiation.. Upregulated expression of Notch1 and its downstream effectors hairy and enhancer of split 1 (Hes1) and Hes1 related with YRPW motif was observed in hesperetin-treated ATC cells. The enhanced luciferase signal also confirmed the functional activity of hesperetin-induced Notch1 signaling. Hesperetin led to a time- and dose-dependent decrease in ATC cell proliferation. The cell-growth inhibition was mainly caused by apoptosis as evidenced by increased levels of cleaved poly ADP ribose polymerase and cleaved caspase-3 as well as decreased survivin. Additionally, hesperetin induced the expression levels of thyrocyte-specific genes including thyroid transcription factor 1 (TTF1), TTF2, paired box gene 8, thyroid stimulating hormone receptor, and sodium/iodide symporter.. Hesperetin activates the Notch1 signaling cascade and suppresses ATC cell proliferation mainly via apoptosis. Hesperetin also induces cell redifferentiation of ATC, which could be useful clinically.

Topics: Adenosine Triphosphatases; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Gene Expression; Hesperidin; Homeodomain Proteins; Humans; Nuclear Proteins; Paired Box Transcription Factors; PAX8 Transcription Factor; Receptor, Notch1; Receptors, Thyrotropin; Signal Transduction; Symporters; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Thyroid Nuclear Factor 1; Transcription Factor HES-1; Transcription Factors

Anaplastic thyroid cancer (ATC) is a very aggressive thyroid gland malignancy with very poor prognosis. It is suspected that the Notch signaling pathway, which is not active in ATC, may have a tumor suppressor function in this neoplasm. However, it remains unknown whether activation of Notch can yield therapeutic efficacies in ATC.. The purpose of this study was to evaluate the effect of chrysin, a potential Notch inducer identified via high-throughput screening, on ATC both in vitro and in vivo.. Chrysin treatment of ATC cells led to a dose-dependent inhibition of cellular growth. Protein and messenger RNA levels of Notch1 and Hes1 (hairy/enhancer of split 1), a downstream Notch1 effector, were both up-regulated with treatment. Luciferase reporter assays incorporating the C promoter-binding factor 1 (CBF1) binding site also confirmed the functional activity of chrysin-induced Notch1. Oral administration of chrysin suppressed the growth of ATC xenografts by an average of 59% compared with the vehicle control group (P = .002). In addition, calculated median time to tumor progression was 11 days for control mice and 21 days for the chrysin treatment group (P = .008). Analysis of chrysin-treated ATC tumors revealed an increase in the active intracellular domain of Notch1 protein. Activation of Notch1 in vivo was associated with the induction of cleaved Poly ADP ribose polymerase (PARP) protein, indicating that the growth inhibition was due to apoptosis.. The novel Notch1 activator chrysin inhibits tumor growth in ATC both in vitro and in vivo. Chrysin could be a promising therapeutic candidate for ATC, and this justifies further clinical studies.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cell Proliferation; Flavonoids; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Mice; Mice, Nude; Poly(ADP-ribose) Polymerases; Receptor, Notch1; Signal Transduction; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Transcription Factor HES-1; Xenograft Model Antitumor Assays