Abram Vaccaro

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

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.

Inactivation of glycogen synthase kinase-3β, a downstream target of the raf-1 pathway, is associated with growth suppression in medullary thyroid cancer cells

Glycogen synthase kinase-3β (GSK-3β) is an important regulator of cell proliferation and survival. Conflicting observations have been reported regarding the regulation of GSK-3β 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-3β. Inactivation of GSK-3β in TT cells with well-known GSK-3β 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-3β 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-3β is a key downstream target of the raf-1 pathway in TT cells. Also, our results show that inactivation of GSK-3β alone is sufficient to inhibit the growth of TT cells both in vitro and in vivo. [Mol Cancer Ther 2007;6(3):1151–8]

ZM336372, A Raf-1 Activator, Causes Suppression of Proliferation in a Human Hepatocellular Carcinoma Cell Line

Hepatocellular carcinoma has been described to exhibit characteristics similar to that of neuroendocrine tumors (NETs). This includes similar anti-neoplastic responses to extracellular signal-regulated kinase (ERK) activation. NET cells and HepG2 cells have both shown growth inhibition with ERK activation. ZM336372, a Raf-1 activating agent, has been shown to cause growth inhibition and suppression of hormone secretion in a neuroendocrine cell line. Here we examine treatment of the HepG2 cell line with ZM336732 to determine if a similar anti-proliferative response will be obtained. HepG2 cells were treated with ZM336372 or solvent (dimethyl sulfoxide). The resulting effect on the proliferation was measured using the 3,4-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Western blot analysis was performed to examine the activation of the Raf-1/mitogen-activated protein kinase kinase/ERK pathway, chromogranin A production, and p21CIP1 level. Growth inhibition was observed with ZM336372 in a dose-dependent fashion. Minimal baseline phosphorylation of ERK 1/2 was observed; however, activation was observed after treatment with ZM336372. Chromogranin A secretion was suppressed due to treatment with ZM336372. A dose-dependent up-regulation of p21CIP1 was observed in response to ZM336372 treatment. ZM336372 causes growth inhibition, suppression of hormone secretion, and up-regulation of cell cycle inhibitors in a human hepatocellular carcinoma cell line, similar to that previously seen in NETs.