Muthusamy Kunnimalaiyaan

Current Management of Medullary Thyroid Cancer

Medullary thyroid cancer accounts for 5%-10% of all thyroid cancers. The majority of medullary thyroid cancers are sporadic, but 20% of cases are a result of a germline mutation in the ret proto-oncogene. Hereditary medullary thyroid cancer can be seen as part of the multiple endocrine neoplasia syndrome type 2A or 2B or as part of familial medullary thyroid cancer. This article discusses the current methods available for the diagnosis and evaluation of a patient with suspected medullary thyroid cancer. The management of medullary thyroid cancer is predominantly surgical excision, consisting of a total thyroidectomy and lymph node dissection. The extent and timing of surgical excision are discussed. Systemic therapeutic options are limited for medullary thyroid cancer, but several therapeutic targets show promise for the development of new therapies in the future.

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]

Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.

Notch 1 signaling is active in ovarian cancer.

OBJECTIVE.: Despite advances in chemotherapy and radical surgery, most advanced stage ovarian cancer patients die from their disease, highlighting the need for the development of novel treatment strategies. The Notch signaling pathway plays an important role in cellular differentiation, proliferation and apoptosis. We hypothesized that the active form of Notch 1, the Notch 1 intracellular domain (NICD), would be overexpressed in ovarian cancer cells and that depletion of NICD would lead to growth reduction. METHODS.: Following institutional review board approval, NICD expression was analyzed in human ovarian cancer specimens as well as the ovarian cancer cell lines OVCAR3, SKOV3, and CaOV3. In addition, the effects of Notch 1 depletion on ovarian cancer cell growth were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay for 6 days following transfection with siRNA against Notch 1. RESULTS.: Western blot analysis revealed abundant NICD expression in all 3 ovarian cancer cell lines, as well as in 16 of 21 (76%) human ovarian cancer samples. Following treatment with Notch 1 siRNA, expression of NICD was greatly reduced in all three cell lines. Furthermore, this depletion of NICD was associated with significant growth inhibition of all three ovarian cancer cell lines. CONCLUSIONS.: NICD was frequently expressed in ovarian cancer cell lines and human ovarian cancer specimens. Importantly, depletion of Notch 1 led to growth inhibition of ovarian cancer cells. These findings support the hypothesis that Notch 1 plays a role in ovarian cancer proliferation, encouraging the investigation of this pathway as a therapeutic target.

ZM336372, a Raf-1 activator, suppresses growth and neuroendocrine hormone levels in carcinoid tumor cells

Neuroendocrine tumors, such as carcinoids, are highly metastatic neoplasms that secrete bioactive hormones resulting in carcinoid syndrome. Few curative treatments exist outside of surgical resection. We have previously shown that activation of the Raf-1 signaling pathway can suppress hormone production in carcinoid tumor cells. In this study, we investigated a novel treatment for carcinoid tumor cell growth based on pharmacologic Raf-1 activation using the compound ZM336372. Treatment of carcinoid tumor cells with ZM336372 resulted in progressive phosphorylation of Raf-1, mitogen-activated protein kinase 1/2, and extracellular signal–regulated kinase 1/2. Importantly, exposure to ZM336372 resulted in a significant reduction of bioactive hormone levels as well as the transcription factor, human achaete-scute homologue-1 in carcinoid tumor cells. Furthermore, treatment with ZM336372 led to a marked suppression of cellular proliferation and induction of the cell cycle inhibitors p21 and p18. In summary, ZM336372 targets both proliferation and palliative issues associated with carcinoid tumor cells, and therefore, warrants further investigation as a possible therapeutic strategy for patients with carcinoid tumors.

The role of human achaete-scute homolog-1 in medullary thyroid cancer cells☆

BACKGROUND Human achaete-scute homolog-1 (hASH1) is a transcription factor that is expressed highly in neuroendocrine tumors such as medullary thyroid cancer (MTC). Thyroid C-cells do not develop in hASH1 knockout mice, which suggests that hASH1 is essential for normal C-cell development. METHODS To determine the effect of raf-1 induction on hASH1 and hormone production, we used an estrogen inducible raf-1 construct in MTC cell line (TT) cells (TT-raf cells). TT or TT-raf cells were treated with control or 1 microM estradiol. After 48 hours, the cells were analyzed for levels of hASH1 and chromogranin A by Western blotting and for calcitonin production by enzyme-linked immunosorbent assay. RESULTS Activation of raf-1 in the TT-raf cells resulted in high levels of phosphorylated MEK and ERK1/2, a morphologic transdifferentiation, and a decrease in chromogranin A and calcitonin levels that are associated with a reduction in hASH1 production. Furthermore, using MEK inhibitors, we demonstrated that these raf-1-mediated changes are dependent on MEK but not ERK1/2 activation. CONCLUSION hASH1 down-regulation by raf-1 in MTC cells is associated with a significant decrease in hormone production. Thus, hASH1 appears to be important in the endocrine phenotype of MTC tumors and may serve as a molecular target for the treatment of patients with MTC.

MicroRNA profiling of benign and malignant pheochromocytomas identifies novel diagnostic and therapeutic targets

MicroRNAs (miRNAs) are small RNAs ( approximately 22 bp) that post-transcriptionally regulate protein expression and are found to be differentially expressed in a number of human cancers. There is increasing evidence to suggest that miRNAs could be useful in cancer diagnosis, prognosis, and therapy. We performed miRNA microarray expression profiling on a cohort of 12 benign and 12 malignant pheochromocytomas and identified a number of differentially expressed miRNAs. These results were validated in a separate cohort of ten benign and ten malignant samples using real-time quantitative RT-PCR; benign samples had a minimum follow-up of at least 2 years. It was found that IGF2 as well as its intronic miR-483-5p was over-expressed, while miR-15a and miR-16 were under-expressed in malignant tumours compared with benign tumours. These miRNAs were found to be diagnostic and prognostic markers for malignant pheochromocytoma. The functional role of miR-15a and miR-16 was investigated in vitro in the rat PC12 pheochromocytoma cell line, and these miRNAs were found to regulate cell proliferation via their effect on cyclin D1 and apoptosis. These data indicate that miRNAs play a pivotal role in the biology of malignant pheochromocytoma, and represent an important class of diagnostic and prognostic biomarkers and therapeutic targets warranting further investigation.

Valproic Acid Activates Notch1 Signaling and Induces Apoptosis in Medullary Thyroid Cancer Cells

Objective:To examine the effects of valproic acid (VPA) on Notch1 expression and cancer cell proliferation in medullary thyroid cancer (MTC) cells. Background:Other than surgery, there are no effective treatments for MTC, a neuroendocrine malignancy that frequently metastasizes. We have previously shown that over-expression of Notch1 in MTC cells inhibits cell growth and hormone production. VPA, a drug long used for the treatment of epilepsy, has recently been identified as a potential Notch1 activator. We hypothesized that VPA might activate Notch1 signaling in MTC cells, with antiproliferative effects. Methods:Human MTC cells were treated with VPA (0–5 mM) and Western blotting was performed to measure levels of Notch1 pathway proteins and neuroendocrine tumor markers. After confirming that VPA is a Notch1 activator in MTC cells, we performed cell proliferation assay. Finally, to determine the mechanism of growth inhibition, we measured protein levels of various markers of apoptosis. Results:Notch1 was absent in MTC cells at baseline. VPA treatment resulted in an increase in both full-length and active Notch1 protein. Notch1 activation with VPA suppressed 2 neuroendocrine tumor markers, ASCL1 and chromogranin A. Importantly, VPA inhibited the growth of MTC cells in a dose-dependent manner. Immunoblot analysis demonstrated caspase activation and poly(ADP-ribose) polymerase cleavage, indicating the induction of apoptosis. Conclusions:VPA activates Notch1 signaling in MTC cells and inhibits their growth by inducing apoptosis. As the safety of VPA in human beings is well established, a clinical trial using this drug to treat patients with advanced MTC could be initiated in the near future.

The Raf-1 pathway: a molecular target for treatment of select neuroendocrine tumors?

Neuroendocrine (NE) tumors such as medullary thyroid cancer, carcinoid, small cell lung cancer and pheochromocytoma are metastatic in nature, and secrete biogenic amines and hormones. In this review, we will discuss the possibility that activation of the Ras/Raf signaling pathway may be a therapeutic target for patients with select NE tumors. In-vitro activation of Raf-1 in NE tumors either by expression of the ectopic catalytic domain of Raf-1 or by a pharmacologic drug, ZM336372, resulted in growth inhibition. In addition, activation of the Ras/Raf pathway led to a significant reduction in NE markers such as serotonin, chromogranin A and calcitonin. These data support development of Raf-1-activating compounds for treatment of patients with NE tumors of selective subtypes.