Medullary Thyroid Carcinoma Cell Line Research Articles

Abstract 2091: Identification of a highly potent, selective and orally available RET inhibitor with antitumor efficacy in RET-dependent tumor models.

Abstract The RET proto-oncogene encodes a receptor tyrosine kinase that is mainly expressed in neural crest-derived tissues where it plays an important role in cell differentiation, growth and survival. Germline activating point mutations of RET are associated with multiple endocrine neoplasia type 2 (MEN2), an inherited cancer syndrome characterized by development of medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid hyperplasia and are present in circa 50% of sporadic cases of MTC. More recently, a chromosomal rearrangement of the RET gene was identified in a subset of lung adenocarcinomas (1-2%) which is reported to result in expression of a fusion protein containing constitutively active RET kinase domain fused to the N-terminal portion of the kinesin KIF5B. We recently presented data on NMS-173, a very potent RET inhibitor characterized by an excellent in vivo activity profile upon i.v. administration. Here we describe the identification and the preclinical characterization of NMS-616, a novel analogue belonging to the same chemical class, suitable for oral administration. NMS-616 is a highly potent (IC50: 2 nM), ATP competitive RET inhibitor, characterized by high selectivity when tested against a panel of more than 50 kinases. NMS-616 potently blocked proliferation of MTC cell lines, such as the TT human cell line, which endogenously harbours constitutively activated RET, concomitant with abrogation of RET autophosphorylation and signaling pathway activation. The compound also inhibited the IL-3 independent proliferation of RET-driven Ba/F3 cells, with down-modulation of RET autophosphorylation and downstream signalling pathways. NMS-616 is characterized by a good in vitro ADME profile and in vivo pharmacokinetic parameters in the mouse, including oral bioavailability. When tested in vivo in a murine subcutaneous xenograft model employing TT cells, NMS-616 displayed dose-dependent tumor growth inhibition following daily oral administration at 50 and 100 mg/Kg, with tumor regression observed at both dose levels. In addition ex vivo analysis demonstrated dose-dependent target modulation following a single administration and the maintenance of RET phosphorylation inhibition for at least 24 hours. Citation Format: Elena Ardini, Patrizia Banfi, Francesca Quartieri, Paolo Polucci, Nilla Avanzi, Dario Ballinari, Laura Mancini, Edward Felder, Daniele Donati, Arturo Galvani, Enrico Pesenti, Antonella Isacchi, Maria Menichincheri. Identification of a highly potent, selective and orally available RET inhibitor with antitumor efficacy in RET-dependent tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2013-2091

Christia vespertilionis plant extracts as novel antiproliferative agent against human neuroendocrine tumor cells

Neuroendocrine tumors respond poorly to radiation and conventional chemotherapy, hence surgical removal of the neoplastic tissue is still the most effective way of treatment. In an attempt to find new therapeutic plant extracts of Christia vespertilionis (CV) their antitumor potential in human medullary thyroid carcinoma (MTC) and human small intestinal neuroendocrine tumor (SI-NET) cell lines were tested. Proliferation and viability were analyzed using cell counting and WST-1 assay. Apoptosis was determined by microscopy, luminescence assays for caspases 3/7, and expression studies of apoptosis-related genes. CV extracts showed antiproliferative and proapoptotic effects in all MTC and SI-NET cell lines, whereby high growth inhibition was observed by treatment with the ethylacetate-extracts (CV-45) in tumor cell lines but not in normal human fibroblasts. Furthermore CV-45 treatment resulted in alterations of gene expression of PDCD5, MTDH and TNFRSF10b in MTC as well as in SI-NET cells. The results indicate that Christia vespertilionis could serve as an anticancer therapeutic for treatment of neuroendocrine tumors.

Mitochondria-Targeted Nitroxide, Mito-CP, Suppresses Medullary Thyroid Carcinoma Cell Survival In Vitro and In Vivo

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. For MTC therapy, the U.S. Food and Drug Administration recently approved vandetanib and cabozantinib, multikinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. We aimed to evaluate mitochondria-targeted carboxy-proxyl (Mito-CP), a mitochondria-targeted redox-sensitive agent, for its tumor-suppressive efficacy against MTC. In vitro cultures of 2 human MTC cell lines, TT and MZ-CRC-1, and TT xenografts in mice were treated with Mito-CP in comparison with vandetanib. The effects on cell survival/death, RET expression, mitochondrial integrity, and oxidative stress were determined. Contrary to vandetanib, Mito-CP induced RET downregulation and strong cytotoxic effects in both cell lines in vitro, including caspase-dependent apoptosis. These effects were accompanied by mitochondrial membrane depolarization, decreased oxygen consumption, and increased oxidative stress in cells. Intriguingly, Mito-CP-induced cell death, but not RET downregulation, was partially inhibited by the reactive oxygen species scavenger, N-acetyl-cysteine, indicating that Mito-CP mediates tumor-suppressive effects via redox-dependent as well as redox-independent mechanisms. Orally administered Mito-CP effectively suppressed TT xenografts in mice, with an efficacy comparable to vandetanib and relatively low toxicity to animals. Our results suggest that Mito-CP can effectively suppress MTC cell growth/survival via a mechanism distinct from vandetanib effects. Mitochondrial targeting may be a potential strategy for MTC therapy.

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.

Anti-proliferative effects of a novel isoflavone derivative in medullary thyroid carcinoma: An in vitro study

Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) α mRNA expression in TT cells was more abundant than ERβ, with a ratio of 48:1. Estradiol-17β (E2) increased DNA synthesis in a dose dependent manner. [3H]-thymidine incorporation was also stimulated by the ERβ agonist DPN and the ERα agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on [3H]-thymidine incorporation could be blocked by the ERβ antagonist PTHPP but not by the ERα antagonist MPP, suggesting that the antiproliferative effect of cD-tBoc on these cells is mediated through ERβ. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.

Translational application of novel withanolides for cancer treatment from Physalis species and other Solanaceae

Natural products have been the most significant source of drugs and drug leads in history. Plant biodiversity provides a resource of unlimited structural diversity for drug lead and molecular probe discovery programs such as those currently underway at the University of Kansas. Technology is gradually overcoming the traditional difficulties encountered in natural products research by improving access to biodiverse resources and ensuring compatibility of samples with high throughput procedures. Our drug lead discovery effort is aimed at uncovering bioactive molecules from plants of the U.S. Great Plains. Approaches utilized in the selection of samples consist of biodiversity-based collections and ethnobotanical information on the medicinal uses in the areas of study. In order to identify antiproliferative compounds, a library of over 1,200 plant extracts was tested for antiproliferative activity against an array of cell lines [melanoma cell lines (B16F10, SKMEL28); human head and neck squamous cell carcinomas (HNSCC) cell lines (MDA1986, JMAR, UM-SCC-2, JHU011); medullary thyroid carcinoma (MTC) cell lines (TT and DRO81–1), and a non-malignant cell line (MRC5)]. In vitro experiments included evaluation of anticancer activity and effects on cell proliferation by MTS assay and trypan blue exclusion. Apoptosis and cell cycle arrest were characterized by annexin V-propidium iodide (PI) flow cytometry and confirmed by Western Blot analysis for activation of caspase 3 and cleavage of PARP as well as cell-cycle specific proteins. Analysis of specific pathways including modulation of Akt, MAP-kinase and RET phosphorylation were evaluated in vitro as well as ex vivo in treated mice using Western blot analysis. Physalis longifolia Nutt. and several related members in the Solanaceae were identified as the most active of the species tested in this study. This led to the discovery of over 30 withanolide-type steroidal lactones, 16 of which are new compounds. In addition to withaferin A (WA), 14 withanolides showed IC50 values in the range between 0.067 and 9.3µM in vitro by MTC with selectivity of 3 to 20 fold compared to normal cells. A mechanistic study showed that WA induced apoptosis and cell death in HNSCC and MTC cells as well as modulation of the cell cycle from G0/G1 arrest to a shift to G2/M and S-phase. Western blot analysis demonstrated that cancer cells treated with WA exhibited inhibition of Akt and caspase-3 expression, and cleavage of PARP. WA was further evaluated in vivo in a metastaic murine model of MTC. All treated animals were alive, showing tumor regression and growth delay without toxicity or weight loss at six weeks post-treatment. Tumor cells treated with WA demonstrated inhibition of total and phospho-RET levels by Western blot analysis in a dose dependent manner (almost complete inhibition with treatment of 5µM WA) as well as potent inhibition of phosphor-ERK and phosphor-Akt levels. WA was shown to be a novel natural-product RET-inhibitor with efficacy in a metastatic murine model of MTC. This investigation constitutes the first report of the antiproliferative activity of WA and other withanolides against MTC and HNSCC. Procurement and screening protocols, isolation and structure determination, lead optimization, and antiproliferative activities of withanolides will be presented.

Preparation and <i>in Vivo</i> Evaluation of Liposomal Everolimus for Lung Carcinoma and Thyroid Carcinoma

Everolimus has demonstrated antitumor efficacy for various cancers as a result of its inhibition of the mammalian target of rapamycin (mTOR) signaling cascade, which activates cell growth and cell proliferation. However, the low water solubility and low bioavailability of everolimus have prevented its clinical development as an anticancer drug. Therefore, to address the unsuitable characteristic of everolimus, we attempted to prepare liposomal everolimus as a viable drug delivery system, and then evaluated the anticancer efficacy of this system against a medullary thyroid carcinoma cell line (TT cells), a breast cancer cell line (MCF-7 cells) and a small lung carcinoma cell line (NCI-H446 cells). The particle size and entrapment efficacy of liposomal everolimus was ca. 80 nm and more than 90%, respectively. Liposomal everolimus showed higher cytotoxicity against NCI-H446 cells compared with TT cells. Against NCI-H446 tumors, significant suppression of the tumor volume was observed in liposomal everolimus-treated mice by intravenous injection, compared with free everolimus-treated mice by intraperitoneal injection, at a dose of 5 mg/kg without body weight loss. This study showed that liposomal everolimus could be a powerful formulation with anticancer efficacy for some cancers.

High-resolution analysis of alterations in medullary thyroid carcinoma genomes

Hereditary and sporadic medullary thyroid carcinoma (MTC) are closely associated with RET proto-oncogene mutations. However, the role of additional changes in the tumor genomes remains unclear. Our objective was the identification of chromosomal regions involved in MTC tumorigenesis and to assess their significance by using MTC-derived cell lines. We used array-CGH (comparative genomic hybridization) to map chromosomal imbalances in 52 primary tumors and ten metastases. Eleven tumors (11/52, 21%) were hereditary and 41 (41/52, 79%) were sporadic. Among the latter, 15 tumors (15/41, 37%) harbored RET mutations. Furthermore, we characterized five MTC cell lines in detail and evaluated the tumorigenicity by severe combined immunodeficiency (SCID)-mouse experiments. Most MTCs had only few copy number changes, and losses of chromosomes 1p, 4q, 19p and 22q were observed most frequently. The number of chromosomal aberrations increased in metastases. Twenty-three percent (12/52) of the primary tumors did not even show any chromosomal gains and losses. We injected three cell lines (two of these were without chromosomal changes and pathogenic RET mutations) into immune deficient SCID mice, and in each case, we observed rapid tumor growth at the injection sites. Our data suggest that MTCs--in contrast to most other tumor entities--do not acquire a multitude of genomic imbalances. SCID mouse experiments performed with chromosomally normal cell lines and without RET mutations suggest that presently unknown submicroscopic genomic changes are sufficient in MTC tumorigenesis.

A water soluble tri-cationic porphyrin–EDTA conjugate induces apoptosis in human neuroendocrine tumor cell lines

In this study, a completely water soluble tri-cationic porphyrin–EDTA conjugate was synthesized. We present data demonstrating the tumoristatic effects of the novel fully water soluble cationic porphyrin TMPy3PhenEDTA-P-Cl4 in the dark, in the medullary thyroid carcinoma cell lines MTC-SK and SHER-I and weaker effects in the small intestinal neuroendocrine tumor cell line KRJ-I. In addition, cytotoxic effects were also studied in normal human fibroblasts that represent normal tissue and the results are compared to the tumor cell lines.

Hyperinsulinemia during oral glucose tolerance test and high normal serum cortisol are associated with increased secretion of calcitonin in normal subjects

Previous studies showed that insulin stimulated directly calcitonin (Ct) secretion in the pig thyroid, while dexamethasone stimulated the production of Ct and Ct mRNA in medullary thyroid carcinoma (MTC) cell lines. The objective of this study was to investigate if hyperinsulinemia during the oral glucose tolerance test (GTT) stimulates Ct secretion in normal subjects as well as to examine the relationship between serum cortisol and Ct. In 26 normal subjects (9 men and 17 women) with detectable basal serum Ct, aged 22-70 yr [51.5±14.6 (mean±SD), median 55.5], we measured serum or plasma Ct, cortisol, ACTH, insulin, and blood glucose before (0 min) and at 30, 60, 90, and 120 min after ingestion of 75 g glucose. During GTT mean serum cortisol increased slightly by 9.3% at 30 min, whereas mean serum insulin increased 9.4-fold, reaching a peak value at 60 min. Median serum Ct increased by 51% (p<0.001) in normal subjects (by 27% in men, p=0.004, and by 44% in women, p<0.001) at 30 min and remained significantly higher thereafter (up to 120 min) when compared to median baseline level. Regression analysis showed that basal serum cortisol, but not basal serum insulin, was correlated with basal serum Ct (p=0.01). Peak concentrations of Ct were also correlated with peak concentrations of cortisol at 30 min (p<0.001) but not at later time points. Serum insulin was correlated with serum Ct at the serum insulin peak level (60 min), and at later time points (90 and 120 min) (p=0.001). Multiple and simple regression analysis showed that calcitonin-AUC (Area Under Curve) values correlated with insulin-AUC (p=0.003), and also with cortisol-AUC (p=0.02) values, the standardized effect of insulin-AUC on Calcitonin-AUC being greater than that of cortisol-AUC. These findings suggest that acute hyperinsulinemia during GTT is very likely associated with increased Ct secretion in normal subjects. Serum cortisol within the physiological range was also correlated with serum Ct under basal conditions, as well as during GTT.

Bystander cytotoxicity in human medullary thyroid carcinoma cells mediated by fusion yeast cytosine deaminase and 5-fluorocytosine

In our work, we have evaluated efficiency of gene-directed enzyme/prodrug therapy (GDEPT) based on combination of fusion yeast cytosine deaminase (yCD) and 5-fluorocytosine (5FC) on model human medullary thyroid carcinoma (MTC) cell line TT. We determined the efficiency of this GDEPT approach in suicide and bystander cytotoxicity induction. We have shown significant bystander effect in vitro and 5FC administration resulted in potent antitumor effect in vivo. Furthermore, we have unraveled high efficiency of cell-mediated GDEPT, when human mesenchymal stromal cells (MSC) were used as delivery vehicles in direct cocultures in vitro. Nevertheless, effector MSC exhibited inhibitory effect on TT cell proliferation and abrogated TT xenotransplant growth in vivo. We suggest that yCD/5FC combination represents another experimental treatment modality to be tested in MTC and our data further support the exploration of MSC antitumor potential for future use in metastatic MTC therapy.

MicroRNA Profiling of Sporadic and Hereditary Medullary Thyroid Cancer Identifies Predictors of Nodal Metastasis, Prognosis, and Potential Therapeutic Targets

While the molecular basis of hereditary medullary thyroid cancer (HMTC) has been well defined, little is known about the molecular pathogenesis of sporadic medullary thyroid cancer (SMTC). In addition, microRNAs (miRNAs) have been shown to be important diagnostic and prognostic markers in cancer but have not been defined in MTC. Our aim was to study the miRNA profile of MTC to identify prognostic biomarkers and potential therapeutic targets. MiRNA microarray profiling was carried out in fresh frozen tissues from patients with SMTC (n = 12) and HMTC (n = 7). Differential expression of three miRNAs was confirmed in a validation cohort of SMTC and HMTC samples (n = 45) using quantitative reverse transcriptase-PCR and correlated with clinical outcomes. The functional role of a selected miRNA was investigated in vitro in the human medullary thyroid carcinoma cell line (TT cells) using cell proliferation assays and Western blotting analysis. MiRs-183 and 375 were overexpressed (P = 0.001; 0.031) and miR-9* was under-expressed (P = 0.011) in SMTC versus HMTC. Overexpression of miRs-183 and 375 in MTC predicted lateral lymph node metastases (P < 0.001; P = 0.001) and was associated with residual disease (P = 0.001; 0.003), distant metastases (P = 0.003; 0.001), and mortality (P = 0.01; 0.011). Knock down of miR-183 expression in the TT cell line induced a significant decrease in the viable cell count and upregulation of the protein LC3B, which is associated with autophagy. Our data indicate that miRNAs play a pivotal role in the biology of MTC and represent an important class of prognostic biomarkers and therapeutic targets warranting further investigation.

Identification of Soluble Candidate Biomarkers of Therapeutic Response to Sunitinib in Medullary Thyroid Carcinoma in Preclinical Models

Medullary thyroid carcinoma (MTC), an aggressive rare tumor due to activating mutations in the proto-oncogene RET, requires new therapeutic strategies. Sunitinib, a potent inhibitor of RET, VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)α/β, has been reported as clinically effective in some patients with advanced MTC. In this study, we examine molecular mechanisms of action of sunitinib and identify candidate soluble biomarkers of response. Both in vitro and in vivo assays, using the human TT RET(C634W) MTC cell line, were done to assess the activity of sunitinib. Kinetic microarray studies were used to analyze molecular pathways modified by sunitinib and to identify candidate biomarkers that were subsequently investigated in the serum of patients. Sunitinib displayed antiproliferative and antiangiogenic activities and inhibited RET autophosphorylation and activation of downstream signaling pathways. We showed that sunitinib treatment induced major changes in the expression of genes involved in tissue invasion and metastasis including vimentin (VIM), urokinase plasminogen (PLAU), tenascin-C (TN-C), SPARC, and CD44. Analyzing downregulated genes, we identified those encoding secreted proteins and, among them, interleukin (IL)-8 was found to be modulated in the serum of xenografted mice under sunitinib treatment. Furthermore, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-β2. Experimental models confirm the clinical efficacy of sunitinib observed in a few studies. Molecular pathways revealed by genomic signatures underline the impact of sunitinib on tissue invasion. Selected soluble candidate biomarkers could be of value for monitoring sunitinib response in metastatic MTC patients.

Octreotide-Targeted Liposomes Loaded with CPT-11 Enhanced Cytotoxicity for the Treatment of Medullary Thyroid Carcinoma

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, but treatment with irinotecan (CPT-11) is limited because of side effects. MTC is known to overexpress the somatostatin receptor subtype 2 (SSTR2). Octreotide (Oct) is a somatostatin analogue that has a high binding affinity for SSTR and can be used as a tumor-targeting ligand. We prepared Oct-targeted liposomes loaded with CPT-11 using Oct-poly (ethylene glycol) (PEG)-lipid and evaluated Oct-mediated association and cytotoxicity of the liposomes with an MTC cell line TT. The association of higher concentrations of modified Oct-targeted liposomes with TT cells was significantly higher than PEGylated liposomes and was significantly inhibited by empty Oct-targeted liposomes but not by free Oct. With exposure for 96 h, the cytotoxicity of Oct-targeted liposomal CPT-11 (IC50: 1.05 ± 0.47 μM) was higher than free CPT-11 (IC50: 3.76 ± 0.61 μM) or PEGylated liposomal CPT-11 (IC50: 3.05 ± 0.28 μM). In addition, empty Oct-targeted liposomes showed significantly higher cytotoxicity than empty nontargeted liposomes at a concentration where free Oct did not show cytotoxicity, suggesting that Oct as a ligand showed cytotoxicity. Moreover, Oct-targeted liposomal CPT-11 led to significantly higher antitumor activity and prolonged the survival time compared with nontargeted liposomal and free CPT-11 at a one-third dose and lower administration times with free CPT-11. These findings indicated that Oct-targeted liposomes loaded with CPT-11 may offer considerable potential for MTC chemotherapy because cytotoxicity of both CPT-11 and Oct was enhanced by effective cellular uptake via SSTR2.

The tyrosine kinase inhibitor ZD6474 blocks proliferation of RET mutant medullary thyroid carcinoma cells

Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.

Abstract 3614: Multi-targeted kinase inhibitor E7080 showed anti-tumor activity against medullary thyroid carcinoma and squamous thyroid carcinoma cell line based on RET and VEGFR2 tyrosine kinase inhibition

Abstract There are about 37,000 and 8,000 new cases of thyroid cancer each year in the US and JP respectively, according to reports of the National Cancer Institute and National Cancer Center in Japan. Thyroid cancer is one of most common cancers after age 30, and its aggressiveness increases significantly in older patients. VEGFR tyrosine kinase inhibitors showed anti-tumor activity against thyroid carcinoma in early clinical study and some of inhibitors have been tested in late stage of clinical trial. E7080, a multi-targeted kinase inhibitor, potently inhibits VEGFR1-3, FGFR1-4 and RET tyrosine kinases and has been tested in several PhII trials including thyroid carcinoma trial. In this study, we report the efficacy of E7080 as an anti-cancer agent for thyroid carcinoma, based on both anti-tumorigenic activity and anti-angiogenic activity. E7080 inhibit RET kinase and VEGFR2 kinase with Ki values of 1.5 and 0.74 nmol/L, respectively, in cell free kinase assays. E7080 inhibited proliferation and phosphorylation of RET as well that of downstream Erk1/2 in medullary thyroid carcinoma (MTC) cell lines, TT and MTC-M, which expressed RET kinase in a dose dependent manner. E7080 showed significantly anti-tumor activity at the dose of 10-100 mg/kg in TT xenograft and 1-100 mg/kg in MTC xenograft without affecting microvessel density, suggesting that anti-tumor activity was caused by anti-tumorigenic activity based on RET kinase inhibition. On the other hand, E7080 did not show anti-proliferative activity against thyroid squamous carcinoma cell line, SW579, which did not expressed RET kinase, even at 10 µmol/L of E7080. While E7080 significantly inhibited in vivo growth of SW579 tumor in nude mice, accompanied by decreased of microvessel density within xenografted tumors at 3 - 100 mg/kg. These results demonstrated that the blockage of RET and VEGFR2 was a promising therapeutic strategy against several types of thyroid carcinoma and E7080, a dual inhibitor of both VEGFR2 and RET tyrosine kinase, is warranted to be examined for thyroid cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3614.

Medullary Thyroid Carcinoma Cell Lines Contain a Self-Renewing CD133+ Population that Is Dependent on RET Activity.

Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET protooncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC. In this study, we sought to identify and characterize cancer stem cell-like cells in MTC. The characterization of stem cell properties was performed using immunostaining, flow cytometry, sphere formation assay, rederivation assay, Western blotting, and quantitative RT-PCR of defined markers of neural stem and progenitor cells. The role of RET activation was assessed through RNA interference knockdown. CD133 positivity was identified by immunostaining patient MTC. Flow cytometry confirmed a subpopulation of CD133+ cells in two MTC cell lines. The CD133+ cells could be expanded by sphere formation assay, passaged multiple times, and expressed neural progenitor markers β-tubulin 3 and glial fibrillary acidic protein. The MZ-CRC-1 cell line, which harbors a M918T RET mutation, had greater CD133+ cell numbers and sphere-forming ability than the TT cell line, which harbors the less active C634W mutation. Sphere formation was more dependent on RET activity than epidermal growth factor or fibroblast growth factor. Our data support the existence of cancer stem-like cells in MTC, which exhibit the features of self-renewal and of multiple lineage differentiation that is dependent on RET receptor activity. These findings may provide new insights to develop more promising therapy for MTC.

Medullary Thyroid Carcinoma Cell Lines Contain a Self-Renewing CD133+Population that Is Dependent onRETActivity

Context: Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET protooncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC. Objective: In this study, we sought to identify and characterize cancer stem cell-like cells in MTC. Main Outcome Measures: The characterization of stem cell properties was performed using immunostaining, flow cytometry, sphere formation assay, rederivation assay, Western blotting, and quantitative RT-PCR of defined markers of neural stem and progenitor cells. The role of RET activation was assessed through RNA interference knockdown. Results: CD133 positivity was identified by immunostaining patient MTC. Flow cytometry confirmed a subpopulation of CD133 cells in two MTC cell lines. The CD133 cells could be expanded by sphere formation assay, passaged multiple times, and expressed neural progenitor markers -tubulin 3 and glial fibrillary acidic protein. The MZ-CRC-1 cell line, which harbors a M918T RET mutation, had greater CD133 cell numbers and sphere-forming ability than the TT cell line, which harbors the less active C634W mutation. Sphere formation was more dependent on RET activity than epidermal growth factor or fibroblast growth factor. Conclusion: Our data support the existence of cancer stem-like cells in MTC, which exhibit the features of self-renewal and of multiple lineage differentiation that is dependent on RET receptor activity. These findings may provide new insights to develop more promising therapy for MTC. Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy Anat Ben-Shlomo, Herbert Schmid, Kolja Wawrowsky, Oxana Pichurin, Erika Hubina, Vera Chesnokova, Ning-Ai Liu, Michael Culler, and Shlomo Melmed (J Clin Endocrinol Metab, published October 9, 2009, 10.1210/jc.2009-1311) ABSTRACT Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933Objective: Pituitary targeted pharmacotherapy for Cushing’s disease is challenging and ineffective. Unlike octreotide and lanreotide, the multisomatostatin receptor (SST) analog pasireotide that exhibits SST5 greater than SST2 binding affinity offers potential for treating Cushing’s disease. Because corticotroph cells express SST5 more abundantly than SST2, pasireotide likely exerts superior corticotroph action mainly through SST5. However, there is no direct evidence for this assumption, and moreover, the ligand effect on corticotroph SST2 is not known. Results: We used AtT20 mouse pituitary corticotroph tumor cells stably overexpressing SST2 or SST5 and TtT/GF mouse pituitary folliculostellate cells stably or transiently expressing SST receptors to examine ligand-receptor activation by SST2and SST5selective agonists. We show that pasireotide was more potent than either octreotide or somatostatin-14 in mouse corticotroph cells. Pasireotide potency is not affected by SST2 abundance, SST2 antagonist treatment, or octreotide cotreatment in SST2overexpressing cells. Pasireotide also does not induce SST2 internalization and attenuates octreotide or SRIF14-induced SST2 internalization only at superphysiological dose ranges. In contrast, octreotide attenuates pasireotide potency in SST5-overexpressing cells. Moreover, short exposure to pasireotide causes prolonged inhibition of forskolin or CRH-induced cAMP accumulation, in contrast to somatostatin-14and SST2-selective agonists that induced postwithdrawal cAMP rebound. Long-term pasireotide signaling effects are enhanced by SST5 overexpression. Conclusion: The results indicate that SST5 determines shortand long-term enhanced pasireotide action in corticotroph cells, whereas the ligand action on SST2 is negligible. Endocrine Reviews, December 2009, 30(7):926–934 edrv.endojournals.org 933

Asymmetrically substituted cationic indole- and fluorene porphyrins inhibit tumor proliferation in small intestinal neuroendocrine tumors and medullary thyroid carcinomas

In this study we demonstrate anticancer activity of novel fully water soluble cationic porphyrins. The two cationic porphyrins 5,10,15-tris(N-methylpyridinium-4-yl)-20-[1-phenyl-4-(3-N-phenylsulfonylindolyl)]-21H,23H-porphyrin chloride (TMPy3PhenIndolprot1P–Cl3) and 5-{5-[2-(9,9-Dimethyl)fluorenyl]-N-methylpyridinium-3-yl}-10,15,20-tris(N-methyl-pyridinium-4-yl)-21H,23H-porphyrin chloride (TMPy3PyFluorenyl1P–Cl4) were prepared and their antiproliferative effects were studied in two human tumor cell lines and a normal human fibroblast cell line. Effects of the novel porphyrin compounds were evaluated in the small intestinal neuroendocrine tumor cell line KRJ-I, the medullary thyroid carcinoma cell line MTC-SK and the normal human fibroblast cell line HF-SAR by cell counting, cell proliferation assays and cell cytotoxicity analyses. TMPy3PhenIndolprot1P–Cl3 and TMPy3PyFluorenyl1P–Cl4 showed antiproliferative effects in the tumor cell lines MTC-SK and KRJ-I; cell viability was decreased and cytotoxic effects were quantified, while no significant alterations of the human fibroblasts were noted. With the advantage of full water solubility and antiproliferative effects in tumor cell lines, the novel porphyrin compounds TMPy3PhenIndolprot1P–Cl3 and TMPy3PyFluorenyl1P–Cl4 could be a new therapeutic option in anticancer treatment.

Medullary Thyroid Carcinoma Cell Lines Contain a Self-Renewing CD133+Population that Is Dependent on Ret Proto-Oncogene Activity

Medullary thyroid carcinoma (MTC) is a cancer of the parafollicular C cells commonly caused by an inherited or acquired RET proto-oncogene mutation. Therapeutic resistance and recurrence of the disease imply the presence of cancer stem cells in MTC. In this study, we sought to identify and characterize cancer stem cell-like cells in MTC. The characterization of stem cell properties was performed using immunostaining, flow cytometry, sphere formation assay, rederivation assay, Western blotting, and quantitative RT-PCR of defined markers of neural stem and progenitor cells. The role of ret proto-oncogene activation was assessed through RNA interference knockdown. CD133 positivity was identified by immunostaining patient MTC. Flow cytometry confirmed a subpopulation of CD133(+) cells in two MTC cell lines. The CD133(+) cells could be expanded by sphere formation assay, passaged multiple times, and expressed neural progenitor markers beta-tubulin 3 and glial fibrillary acidic protein. The MZ-CRC-1 cell line, which harbors a M918T RET mutation, had greater CD133(+) cell numbers and sphere-forming ability than the TT cell line, which harbors the less active C634W mutation. Sphere formation was more dependent on ret proto-oncogene activity than epidermal growth factor or fibroblast growth factor. Our data support the existence of cancer stem-like cells in MTC, which exhibit the features of self-renewal and of multiple lineage differentiation that is dependent on ret proto-oncogene receptor activity. These findings may provide new insights to develop more promising therapy for MTC.