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...

Carcinoid tumors are neuroendocrine malignancies that frequently metastasize and secrete hormones that cause debilitating symptoms in patients. In this study we report the effects of valproic acid (VPA), a drug long used for the treatment of epilepsy, on the growth and neuroendocrine phenotype of human carcinoid cancer cells. VPA treatment of gastrointestinal and pulmonary carcinoid cells resulted in a dose-dependent inhibition of cancer cell growth. Western blot analysis revealed degradation of cyclin D1 and an increase in cyclin-dependent kinases p21 and p27 with VPA treatment. Flow cytometry confirmed that the mechanism of VPA-induced growth inhibition is G 1 phase cell cycle arrest. Furthermore, VPA suppressed expression of the neuroendocrine tumor marker chromogranin A. In addition to these effects, VPA also increased levels of full- Disclosure of potential conflicts of interest is found at the end of this article.

Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging (124I) or molecular radiotherapeutic (131I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. 131I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with 124I-CLR1404 or 131I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular 124I-CLR1404 tumor imaging for planning 131I-CLR1404 therapy.

Glycogen synthase kinase-3B (GSK-3B) is an important regulator of cell proliferation and survival. Conflicting observations have been reported regarding the regulation of GSK-3B 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-3B. Inactivation of GSK-3B in TT cells with well-known GSK-3B 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-3B 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-3B is a key downstream target of the raf-1 pathway in TT cells. Also, our results show that inactivation of GSK-3B alone is sufficient to inhibit the growth of TT cells both in vitro and in vivo.

Background-Surgical resection is the only curative treatment for patients with pheochromocytomas, paragangliomas, and other catecholamine-producing tumors. Activation of glycogen synthase kinase 3β (GSK3β) is thought to promote tumor growth and neuroendocrine (NE) peptide secretion in NE tumors. Thus, we hypothesized that inhibition of this signaling pathway with lithium chloride (LiCl), a well-known GSK3β inhibitor, could be a potential therapeutic strategy to control tumor growth and hormone production.