Overexpression of the NOTCH1 Intracellular Domain Inhibits Cell Proliferation and Alters the Neuroendocrine Phenotype of Medullary Thyroid Cancer Cells

Abstract

The role of NOTCH1 as an oncogene or tumor suppressor appears to be cell type-specific. Medullary thyroid cancer (MTC) cells characteristically express the transcription factor ASCL1 (achaete-scute complex-like 1) as well as high levels of the neuroendocrine (NE) markers calcitonin and chromogranin A (CgA). In this study, we show that the active NOTCH1 intracellular domain is absent in human MTC tumor tissue samples and MTC-TT cells. To determine the effects of NOTCH1 expression, we created a doxycycline-inducible NOTCH1 intracellular domain in MTC cells (TT-NOTCH cells). Treatment of TT-NOTCH cells with doxycycline led to dose-dependent induction of NOTCH1 protein with corresponding decreases in ASCL1 protein and NE hormones. ASCL1 promoter-reporter assay and Northern analysis revealed that ASCL1 reduction by NOTCH1 activation is predominantly via silencing of ASCL1 gene transcription. Overexpression of ASCL1 in MTC cells indicated that CgA expression is highly dependent on the levels of ASCL1. This was further confirmed by experiments using small interfering RNA against ASCL1, in which reduction in ASCL1 led to reduction in both CgA and calcitonin. Furthermore, we demonstrate that NOTCH1 signaling activation leads to ERK1/2 phosphorylation, but that reduction in NE markers is independent of ERK1/2 activation. Activation of NOTCH1 resulted in significant MTC cell growth inhibition. Notably, reduction in MTC cell growth was dependent on the level of NOTCH1 protein present. Moreover, no increase in growth upon expression of ASCL1 in NOTCH1-activated cells was observed, indicating that the growth suppression observed upon NOTCH1 activation is independent of ASCL1 reduction. Mechanistically, we show that MTC cell growth inhibition by NOTCH1 is mediated by cell cycle arrest associated with up-regulation of p21. Medullary thyroid cancer (MTC)2 is a neuroendocrine (NE) tumor derived from the calcitonin-producing C-cells of the thyroid gland and accounts for 3-5% of cases of thyroid cancer (1, 2). The only curative therapy for patients with MTC is surgical resection. Eighty percent of all MTCs are sporadic in nature, and the remaining 20% are familial and caused by germ line mutations in the RET proto-oncogene (2, 3). Although development of RET gene testing has allowed for early prophylactic thyroidectomy for patients with familial MTC, the majority of patients with sporadic MTC have persistent or recurrent disease after surgery because the natural history of MTC is characterized by early metastasis. Understanding the molecular pathways that control MTC and C-cell development and proliferation is essential for the development of novel therapies for patients with advanced MTC.Like other NE tumors, MTC cells secrete various hormones and NE markers such as calcitonin and chromogranin A (CgA) (4). In addition, MTC cells express high levels of ASCL1 (achaete-scute complex-like 1, also known as human ASH1 (achaete-scute homolog-1)), an evolutionarily conserved basic helix-loop-helix transcription factor that seems to be lim...

Expression of Notch-1 and alteration of the E-cadherin/β-catenin cell adhesion complex are observed in primary cutaneous neuroendocrine carcinoma (Merkel cell carcinoma)

“…4 Recent studies demonstrated that Notch-1 signaling is minimal or absent in carcinoids, medullary thyroid cancer and small-cell lung cancer, supporting the hypothesis of a tumorsuppressor function of Notch-1 in neuroendocrine tumors. [5][6][7][8] Finally, in vitro experiments have shown that exogenous activation of Notch-1 signaling results in tumor suppression and in a significant reduction in neuroendocrine markers such as chromogranin A, serotonin and calcitonin. 8 Primary neuroendocrine carcinoma of the skin or Merkel cell carcinoma is a rare and often aggressive malignant primary cutaneous cancer.…”

A chimeric fusion of the hASH1 and EZH2 promoters mediates high and specific reporter and suicide gene expression and cytotoxicity in small cell lung cancer cells

“…hASH1EZH2 promoter for SCLC gene therapy TT Poulsen et al transcription when introduced into SCLC and MTC cells, 17,20 and we therefore evaluated the expression of these proteins in the SCLC and control cell lines by western blot analysis (Figure 3a). HES-1 protein was undetectable in MARH24 or NCI417, while DMS273 expressed high levels.…”

“…18 Furthermore, the HES-1 upstream activator Notch1 has been found to potently decrease hASH1-mRNA levels in SCLC cells in a HES-1 independent manner and direct transcriptional silencing of the hASH1 gene by exogenous Notch1 expression was recently demonstrated in medullary thyroid cancer (MTC) cells. 19,20 The gene enhancer of zeste homolog 2 (EZH2) is highly expressed in a number of malignancies including breast cancer and metastatic prostate cancer. [21][22][23] and in many proliferating cells and cell lines.…”

Novel Approaches in Anaplastic Thyroid Cancer Therapy

“…In addition to RAS, BRAF, RET/PTC, and p53, the Notch family receptors and ligands were recently found to be involved in the proliferation and differentiation of thyroid cancer [17,18]. We will cover the main pathways to date that are known to contribute to thyroid oncogenesis, tumorigenesis, and dedifferentiation, including RET-RAS-RAF, Notch1, and histone deacetylase (HDAC).…”

“…Depending on the cell type, Notch can function as either an oncogene or a tumor suppressor [18,[57][58][59][60][61][62]. Ferretti et al [17] first demonstrated that the expression of Notch was decreased in DTC and further in ATC when compared with normal thyroid tissue.…”