ABSTRACT
Notch1-3 are transmembrane receptors that appear to be absent in medullary thyroid cancer (MTC). Previous research has shown that induction of Notch1 has a tumor-suppressor effect in MTC cell lines, but little is known about the biologic consequences of Notch3 activation for the progression of the disease. We elucidate the role of Notch3 in MTC by genetic (doxycycline-inducible Notch3 intracellular domain) and pharmacologic [AB3, novel histone deacetylase (HDAC) inhibitor] approaches. We find that overexpression of Notch3 leads to the dose-dependent reduction of neuroendocrine tumor markers. In addition, Notch3 activity is required to suppress MTC cell proliferation, and the extent of growth repression depends on the amount of Notch3 protein expressed. Moreover, activation of Notch3 induces apoptosis. The translational significance of this finding is highlighted by our observation that MTC tumors lack active Notch3 protein and reinstitution of this isoform could be a therapeutic strategy to treat patients with MTC. We demonstrate, for the first time, that overexpression of Notch3 in MTC cells can alter malignant neuroendocrine phenotype in both in vitro and in vivo models. In addition, our study provides a strong rationale for using Notch3 as a therapeutic target to provide novel pharmacologic treatment options for MTC.
Subject(s)
Receptors, Notch/genetics , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Tumor Suppressor Proteins/metabolism , Animals , Apoptosis/drug effects , Carcinoma, Neuroendocrine , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Inhibitory Concentration 50 , Male , Mice, Nude , Neurosecretory Systems/metabolism , Neurosecretory Systems/pathology , Phenotype , Receptor, Notch3 , Receptors, Notch/metabolism , Signal Transduction/drug effects , Small Molecule Libraries/pharmacologyABSTRACT
Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC.
