Cancer Research Targets Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 6956, July 15, 2007. doi: 10.1158/0008-5472.CAN-06-4605
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Akeno-Stuart, N.
Right arrow Articles by Fagin, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Akeno-Stuart, N.
Right arrow Articles by Fagin, J. A.

Experimental Therapeutics, Molecular Targets, and Chemical Biology

The RET Kinase Inhibitor NVP-AST487 Blocks Growth and Calcitonin Gene Expression through Distinct Mechanisms in Medullary Thyroid Cancer Cells

Nagako Akeno-Stuart1, Michelle Croyle1, Jeffrey A. Knauf1, Roberta Malaguarnera1, Donata Vitagliano2, Massimo Santoro2, Christine Stephan3, Konstantina Grosios3, Markus Wartmann3, Robert Cozens3, Giorgio Caravatti3, Doriano Fabbro3, Heidi A. Lane3 and James A. Fagin1

1 Division of Endocrinology and Metabolism, University of Cincinnati, Cincinnati, Ohio; 2 Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, University Federico II, Naples, Italy; and 3 Novartis Institutes for BioMedical Research, Oncology Research, Basel, Switzerland

Requests for reprints: James A. Fagin, Department of Medicine and Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 296, New York, NY 10021. E-mail: faginj{at}mskcc.org or Heidi A. Lane, Novartis Institutes for BioMedical Research, Oncology WKL-125.13.17, Klybeckstrasse 141, CH-4057 Basel, Switzerland. E-mail: heidi.lane{at}novartis.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The RET kinase has emerged as a promising target for the therapy of medullary thyroid cancers (MTC) and of a subset of papillary thyroid cancers. NVP-AST487, a N,N'-diphenyl urea with an IC50 of 0.88 µmol/L on RET kinase, inhibited RET autophosphorylation and activation of downstream effectors, and potently inhibited the growth of human thyroid cancer cell lines with activating mutations of RET but not of lines without RET mutations. NVP-AST487 induced a dose-dependent growth inhibition of xenografts of NIH3T3 cells expressing oncogenic RET, and of the MTC cell line TT in nude mice. MTCs secrete calcitonin, a useful indicator of tumor burden. Human plasma calcitonin levels derived from the TT cell xenografts were inhibited shortly after treatment, when tumor volume was still unchanged, indicating that the effects of RET kinase inhibition on calcitonin secretion were temporally dissociated from its tumor-inhibitory properties. Accordingly, NVP-AST487 inhibited calcitonin gene expression in vitro in TT cells, in part, through decreased gene transcription. These data point to a previously unknown physiologic role of RET signaling on calcitonin gene expression. Indeed, the RET ligands persephin and GDNF robustly stimulated calcitonin mRNA, which was blocked by pretreatment with NVP-AST487. Antagonists of RET kinase activity in patients with MTC may result in effects on plasma calcitonin that are either disproportionate or dissociated from the effects on tumor burden, because RET kinase mediates a physiologic pathway controlling calcitonin secretion. The role of traditional tumor biomarkers may need to be reassessed as targeted therapies designed against oncoproteins with key roles in pathogenesis are implemented. [Cancer Res 2007;67(14):6956–64]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The RET gene encodes the signaling subunit of a receptor complex for ligands of the glial-derived neurotrophic factor family (1), which in turn, bind to a family of GFR{alpha} (GDNF family receptor {alpha}) coreceptors, consisting of four glycosyl-phosphatidyl-inositol–anchored proteins, GFR{alpha}1–4, which form a complex with RET tyrosine kinase. GFR{alpha}1–4 serve as preferential receptors for GDNF, neurterin, artemin, and persephin, respectively (2). RET is expressed in the developing central and peripheral nervous systems and in the renal excretory system (3, 4). At days 8.5 to 9.5, RET expression is restricted to neural crest cells, including those that later give rise to the calcitonin-secreting C cells of the developing thyroid gland (4). Among endocrine tissues, RET is also expressed in the adrenal medulla (5).

Distinct germ line–activating mutations of RET confer predisposition to the major variants of multiple endocrine neoplasia type 2 (MEN2). MEN2A is primarily associated with mutations in Cys residues in the extracellular domain of RET, leading to ligand-independent covalent dimerization and activation of the receptor, and to predisposition to medullary thyroid cancers (MTC), pheochromocytomas, and parathyroid hyperplasia. MEN2B is associated with kinase domain mutations of RET, which results in high penetrance of MTC with onset at an early age, neural abnormalities of the gastrointestinal tract, and mucosal neuromas (6). In addition to its role in the tumorigenesis of C cells and adrenal medullary cells, the RET gene is activated by somatic recombination events in papillary thyroid cancer (RET/PTC). The function of RET has been studied extensively in vivo, and in various cell types in vitro. RET is required for renal organogenesis and enteric neurogenic development (7). RET signaling is also required for the migration of sympathoadrenal neural crest cells, primarily in response to the ligand artemin (8). Inappropriate RET activation by mutations promotes cell survival in pheochromocytoma cells (9), TSH-independent growth in thyroid follicular cells (10), and cell proliferation and transformation in NIH3T3 cells (11).

The pathogenetic role of RET mutations in MTC and PTC has generated interest in developing small molecule antagonists of RET kinase activity. Molecules of various chemical structures have been found to be effective RET kinase inhibitors (12). The pyrazolo-pyrimidines (PP1 and PP2; ref. 13), the indocarbazole derivatives, CEP-701 and CEP-751 (14), and the 2-indolinone RPI-1 (15) exert growth-inhibitory effects on the human MTC cell line TT in vitro and, for many of these compounds, in MTC xenografts. The quinazoline ZD6474 is also a potent RET inhibitor (16), and is presently in clinical trials for patients with familial MTC associated with germ line RET mutations.

Here, we report on the effects of a novel compound, NVP-AST487, a N,N'-diphenyl urea, which exhibits potent RET-inhibitory activity in vitro and in vivo. Because plasma levels of calcitonin are a useful marker of tumor burden in patients with MTC, we monitored calcitonin levels in mice with MTC xenografts treated with NVP-AST487 and observed that they dropped precipitously prior to any effects on tumor mass. This led to the discovery of a novel mechanism of regulation of calcitonin gene expression, controlled by the ligand persephin via interaction with the RET-GFR{alpha} coreceptor complex, and subject to direct inhibition by RET kinase antagonists. Our findings also illustrate a potential caveat of targeted therapies, which may independently modulate the expression of tumor markers and compromise their usefulness as indicators of tumor burden.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. NVP-AST487-NX, a protein tyrosine kinase inhibitor with the structure 1-[4-(4-ethyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenyl]-3-[4-(6-methylamino-pyrimidin-4-yloxy)-phenyl]-urea was synthesized at Novartis Pharma AG. For in vitro experiments, it was resuspended in DMSO, and diluted with PBS. ZD6474 was kindly provided by Dr. Anderson Ryan (Astra-Zeneca, Macclesfield, United Kingdom). The rat calcitonin immunoradiometric (IRMA) kit was purchased from Immutopics, and the human calcitonin ELISA was from MD Biosciences. Chloramphenicol acetyltransferase (CAT) ELISA was from Roche, the Steady-Glo luciferase assay system was from Promega, whereas actinomycin D was from U.S. Biochemical. GDNF, persephin, and recombinant rat GFR{alpha}1/Fc were from R&D Systems, Inc.

Cell lines. The human thyroid carcinoma cell lines NPA, ARO, FRO, and WRO were maintained in RPMI 1640 supplemented with 10% FCS. The human papillary thyroid cancer cell line TPC-1 was maintained in DMEM with 10% FCS. The human medullary carcinoma cell line TT was maintained in DMEM/Ham's F12 medium supplemented with 10% FCS. MTC-M cells, a murine MTC line mouse thyroid cell line, was grown in suspension in RPMI 1640 supplemented with 15% horse serum and 5% FCS. PCCL3-RET/PTC3 cells are derived from the well differentiated, nontransformed rat thyroid cell line PCCL3 and conditionally express RET/PTC3 in a doxycycline-dependent manner, and were propagated in H4 complete medium. NIH3T3-RETC634W stably express the indicated activating point mutated RET oncoprotein.

Preparation of enzymes and kinase assays. Glutathione S-transferase (GST)–fused kinase domains were expressed in baculovirus and purified over glutathione-sepharose. Kinase activity was tested by measuring the phosphorylation of a synthetic substrate [poly(Glu, Tyr)], by purified GST-fusion kinase domains of the respective protein kinase in the presence of radiolabeled ATP; the ATP concentrations used were optimized within the Km range for the individual kinases. Briefly, each kinase was incubated under optimized buffer conditions in 20 mmol/L of Tris-HCl (pH 7.5), 1 to 3 mmol/L of MnCl2, 3 to 10 mmol/L of MgCl2, 10 µmol/L of Na3VO4, 1 mmol/L of DTT, 0.2 µCi [–33P]ATP, 1 to 8 µmol/L of ATP, 3 to 8 µg/mL of poly(Glu/Tyr, 4:1), and 1% DMSO in a total volume of 30 µL in the presence or absence of NVP-AST487 for 10 min at ambient temperature. Reactions were terminated by adding 10 µL of 250 mmol/L EDTA, and the reaction mixture was transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore). Filters were washed (0.5% H3PO4), soaked in ethanol, dried and counted in a liquid scintillation counter. IC50s for NVP-AST487 were calculated by linear regression analysis of the percentage inhibition.

Athymic mouse TT cell xenografts. Female athymic nude mice were kept under optimized hygienic conditions (maximum of 10 mice per Makrolon type III cage) with free access to food and water. Tumors were established by s.c. injection of 1 x 106 and 5 x 106 of NIH3T3-RETC634W and TT cells, respectively, in 100 µL of HBSS per mouse. Treatable tumors, i.e., mean tumor volume of ~100 mm3, developed within 10 days of NIH3T3-RETC634W cell injection, and within 20 days of TT cell injection. NVP-AST487 was given p.o., once daily by gavage. The compound was formulated by dissolving the appropriate amount of powder in N-methylpyrrolidone/PEG300 (1:10 v/v). The mice were randomized into four treatment groups of eight mice each. The first three groups received daily oral administrations of NVP-AST487 at 50, 30, and 10 mg/kg, respectively, for 3 weeks. The fourth group received treatment with vehicle. Tumor growth and body weights were monitored twice weekly. Tumor volumes were determined according to the formula: length x diameter2 x {pi}/6. Tumors were collected and frozen in liquid nitrogen at the end of the efficacy study, 6 h after the last administration.

Western blotting. Cells were lysed in a buffer containing 20 mmol/L of Tris-HCl (pH 7.5), 3 mmol/L of EDTA, 150 mmol/L of NaCl, 0.5% Triton-X, 50 mmol/L of NaF, 10 mmol/L of Na-PPi, 2 mmol/L of phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L of sodium orthovanadate, and protease inhibitor cocktail (Sigma). The tumor tissues were homogenized at 4°C for 15 s in 10 volumes of lysis buffer using an Ultra-Turrax (Model T25). The lysis buffer was composed of 50 mmol/L of Tris-HCl (pH 7.5), 5 mmol/L of EGTA, 1% Triton X-100, 150 mmol/L of NaCl, 1 mmol/L of PMSF, 80 µg/mL of aprotinin, 50 µg/mL of leupeptin, and 200 µmol/L of sodium orthovanadate. Following 30 min of incubation on ice, the lysates were cleared by centrifugation and the supernatant collected and stored at –70°C. Before freezing, a sample aliquot was diluted at 1:2,000 in water for determination of the protein concentration with a commercially available protein assay kit using bovine serum albumin as standard (Pierce).

Sixty micrograms of each sample was separated by SDS-PAGE and blotted onto polyvinylidene difluoride or nitrocellulose membrane (Amersham Biosciences). The following primary antibodies were used: anti–phospho-ERK, anti-ERK1 K-23, anti-PLC{gamma} (Santa Cruz Biotechnology), anti-RET (Yuri Nikiforov, University of Cincinnati, Cincinnati, OH), or polyclonal rabbit anti-RET (C-19; Santa Cruz Biotechnology), anti-p905-RET and anti-p783 PLC{gamma} (Cell Signaling). The antigen-antibody complexes were visualized using horseradish peroxidase–conjugated anti-mouse or rabbit IgG antibody (Santa Cruz Biotechnology) and enhanced chemiluminescence system (Amersham Biosciences). The membrane was stripped using a Restore Western blot Stripping Buffer (Pierce), and reprobed several times. For quantitation, gel images were captured using a Kodak image station and band density was determined using a one-dimensional image analysis software.

RET immunoprecipitation. Washed cells were lysed in ice-cold buffer containing 50 mmol/L of HEPES (pH 7.4), 150 mmol/L of NaCl, 25 mmol/L of ß-glycerophosphate, 25 mmol/L of NaF, 5 mmol/L of EGTA, 1 mmol/L of EDTA, 15 mmol/L of Na-PPi, 2 mmol/L of sodium orthovanadate, 10 mmol/L of sodium molybdate, leupeptin (10 µg/mL), aprotinin (10 µg/mL), 1 mmol/L of DTT, 1 mmol/L of PMSF, and 1% NP40. Protease inhibitors and NP40 were purchased from Sigma. Extracts were homogenized, cleared by centrifugation, aliquoted, and frozen at –70°C. Total protein (500 µg; Bio-Rad protein assay reagent) was immunoprecipitated with 1 µg of RET-specific antibody (C-19), and the immunoprecipitates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride. Total tyrosine phosphorylation was assessed with the antiphosphotyrosine antibody 4G10 (Cell Signaling). The membrane was stripped and reprobed with the antiphosphotyrosine 1062 RET antibody (Santa Cruz Biotechnology). RET protein levels were controlled by direct Western blots using C-19. Decorated proteins were revealed as above.

Northern blot analysis and real-time reverse transcription-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen Corporation) and 6 to 10 µg RNA samples were resolved by formaldehyde agarose gel electrophoresis in HEPES buffer, transferred to nylon membranes, cross-linked by UV irradiation, and hybridized. The following probes were used: mouse calcitonin 0.8 kb cDNA probe, a 0.9 kb human calcitonin cDNA probe, a 682 bp human carcinoembryogenic antigen (CEA) cDNA constructed using the following primers: (5'-TCCTCTATGGCCCAGACGAC-3' and 5'-TGTGTGTGTTGCTGCGGTATC-3') or a 1.1 kb human cyclin D1 cDNA. Probes were labeled by random priming using the Prime II kit (Stratagene). After washing, signal intensity was quantitated by phosphorimaging and analyzed using ImageQuant (Molecular Dynamics, Inc.) image analysis software. Blots were standardized by either ethidium bromide staining of 18S and 28S rRNAs, or by hybridization with a mouse ß-actin cDNA probe. Additional experiments looking at the effects of NVP-AST487, 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)-pyrazolo-[3,4-d]-pyrimidine (PP2) or ZD6474 on calcitonin or calcitonin gene–related peptide (CGRP) mRNA levels were done by real-time reverse transcription-PCR. Primer set 1 amplified a fragment bracketing exons 2 and 3, which is common to both calcitonin and CGRP. Primer set 2 amplified a fragment spanning exons 3 and 4, which was specific to CGRP. Primers are available upon request.

Sequencing of mouse RET cDNA from MTC-M cells. The sequence of RET cDNA from MTC-M cells was determined by reverse transcription-PCR, using overlapping primers spanning nucleotides 139 to 3,265. The only substitution leading to an amino acid change was a TCC (serine) to TTC (phenylalanine) at codon 520. This is likely a polymorphism because some of the mouse RET sequences present in the Genbank contain a serine (accession no. NM_009050, X67812, or 19713) whereas others (accession no. BC059012, AF209436, AY326397, or NC_000072) contain a phenylalanine.

Calcitonin immunoassays. Human calcitonin was assayed from serum of athymic mice with TT cell tumor xenografts and from TT cell–conditioned media by ELISA (MD Biosciences). Fluorescence was measured in a FLUOstar OPTIMA Platereader (BMG). Calcitonin levels in the plasma of wild-type mice were measured in an IRMA kit according to the manufacturer's instructions (Immutopics).

Transient transfection assays. Plasmid DNAs were prepared using commercial kits (Bio-Rad). TT cells were cultured to 70% confluence in six-well plates. The CAT reporter plasmids, pCT132CAT and pCT252CAT, were a gift from Dr. Barry Nelkin (Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD), and pGL3-control was purchased from Promega. For each well, 2.4 µg of plasmid DNA (2 µg of reporter and 0.4 µg of pGL3-control) were mixed together and incubated at room temperature for 15 min with 20 µL of LipofectAMINE Plus and 5.0 µL of LipofectAMINE (Invitrogen). Plates were washed with serum-free medium and then incubated at 37°C for 5 h with the transfection solution, after which time, medium was changed to growth medium. Cells were then left untreated or were treated with 100 nmol/L of NVP-AST487 for 48 h. CAT assays were carried out using CAT ELISA and measured by FLUOstar OPTIMA Plate Reader. Luciferase assays were carried out using steady-Glo luciferase assay system and activities were measured by Top count (Packard). The CAT activity (mean ± SE) was calculated as picograms per microgram of protein and normalized by luciferase activity. All experiments were done by triplicate and repeated thrice. The protein concentration was measured using the Coomassie Plus protein assay reagent (Pierce Chemical, Co.).

Statistical analyses. Data were compared by t test or by ANOVA followed by Bonferroni t test. Significance was defined as P < 0.05. IC50 was calculated with PRISM software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro selectivity of NVP-AST487 and oral bioavailability. NVP-AST487, a novel RET tyrosine kinase inhibitor of the N,N'-diphenyl urea class, inhibited the ICD of the RET kinase with an IC50 of 0.88 µmol/L (Table 1 ). A number of other kinases were also similarly inhibited by NVP-AST487 in the in vitro kinase assays, including KDR, Flt-4, Flt-3, c-Kit, and c-Abl. Additionally, data derived from a panel of Baf3 murine pro–B cell lymphoma lines rendered growth factor–independent by transduction with various activated tyrosine kinases, suggested cellular specificity for RET-driven proliferation (IC50 for PTC3-RET–driven Baf3 cells, 34 ± 4 nmol/L), with activity against FLT3 as well, and to a lesser extent, Bcr-ABL–dependent proliferation (data not shown). After a single oral administration of 15 mg/kg of NVP-AST487 to OF1 mice, a mean peak plasma level (Cmax) of 0.505 ± 0.078 µmol/L SE was achieved after 0.5 h. Similar levels of NVP-AST487 were found in the plasma of mice up to 6 h after oral administration, with a Clast of 0.021 ± 0.004 µmol/L at 24 h. The oral bioavailability was calculated to be 9.7% with a t1/2 terminal elimination of 1.5 h.


View this table:
[in this window]
[in a new window]

  		Table 1. In vitro kinase activity of NVP-AST487

 
NVP-AST487 inhibits RET autophosphorylation and activation of PLC{gamma} and ERK. We examined the effects of NVP-AST487 on RET autophosphorylation and signaling in PC-RET/PTC3 cells and in TT cells. PC-RET/PTC3 thyroid cells express oncogenic RET/PTC3 in a doxycycline-inducible manner. Induction of RET/PTC3 by treatment with doxycycline for 24 h was associated with autophosphorylation at RETY905, which was decreased by NVP-AST487 in a dose-dependent manner (5–60 nmol/L). A similar profile of inhibitory activity was seen on pPLC{gamma} and pERK (Fig. 1A ). The effects of NVP-AST487 were also examined in TT cells, which harbor an endogenous activating point mutation of RET (RETC634W). NVP-AST487 inhibited autophosphorylation in RET immunoprecipitates as determined by Western blotting with antibodies to pRETY1062 or to total phosphotyrosine (Fig. 1B). The potency of NVP-AST487 on RET activity in PCCL3 or TT cells was >20-fold greater than that observed in the in vitro kinase assays.


Figure 1
View larger version (9K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. NVP-AST487 inhibits RET autophosphorylation and downstream signaling. A, Western blots of PC-PTC3 lysates from cells incubated with or without doxycycline for 24 h in the presence or absence of the indicated concentration of NVP-AST487. Both compounds were added simultaneously. Incubation with 1 µg/mL of doxycycline results in the induction of RET/PTC3 expression and autophosphorylation of RET as determined by a phosphospecific antibody to RETY905. Preincubation with NVP-AST487 induced a dose-dependent reduction in phosphorylation at RETY905, as well as of PLC{gamma} and ERK. B, Western blots of RET immunoprecipitates from TT cell extracts derived from cells incubated with the indicated concentrations of NVP-AST487 for 90 min. Blots were probed for total phosphotyrosine content (p-Tyrosine) or phosphorylation of RET on tyrosine 1062 [p-RET(Y1062)]. RET protein levels were analyzed by direct Western blot (RET). Incubation with NVP-AST487 induced a dose-dependent reduction in RET phosphorylation.

 
NVP-AST487 inhibits the growth of human thyroid cancer cell lines with RET, but not BRAF mutations. We tested the effects of NVP-AST487 on the growth of human thyroid cancer cell lines known to harbor activating mutations of the following oncogenes: (a) RET: TPC-1 (RET/PTC1) and TT (RETC634W; ref. 17); (b) BRAFT1796A: NPA, ARO, and FRO (18, 19). We also tested its effects on WRO, which have no known mutation of TK receptors, RAS or BRAF. IC50 for growth for TT and TPC-1 cells was 7.2 and ~5 nmol/L, respectively (Fig. 2A ). By contrast, the compound had no effect on the growth of cell lines with the BRAF mutation or on WRO cells (Fig. 2B). These data support the selectivity of the compound for RET (relative to RAF), and suggest that the growth of PTC and MTC cells requires RET kinase activity.


Figure 2
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. NVP-AST487 inhibits the growth of thyroid cancer cell lines with activating mutations of RET, but not of cells without RET mutations. Dose-dependent effects of NVP-AST487 on the growth of TPC-1 and TT cells (A). Cells were plated 24 h prior to addition of the indicated concentrations of the kinase inhibitor. Data represent the cell counts after subtraction of the plating number. Growth curves of TPC-1 and NPA, ARO, FRO, and WRO cells (B) treated with or without 100 nmol/L of NVP-AST487.

 
Effects of NVP-AST487 on the growth of cell lines expressing oncogenic RET in vivo. NVP-AST487 given p.o. evoked a dose-dependent inhibition of growth of NIH3T3-RETC634W xenografts, with doses >30 mg/kg/d causing significant reductions in tumor size (Fig. 3A ). The effects of the compound on RET expression and phosphorylation in tumor extracts was analyzed 6 h following the final treatment (Fig. 3B). Reductions in tumor RET phosphorylation in NVP-AST487–treated animals were clearly seen, particularly at doses ≥30 mg/kg. Interestingly, there was also a dose-dependent decrease of RET expression, with one of three tumors analyzed in the 30 mg/kg group and three of three tumors in the 50 mg/kg group showing a dramatic reduction in RET protein levels.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. NVP-AST487 inhibits the growth of NIH3T3-RETC634W xenografts in athymic nude mice. A, female athymic nude mice were s.c. injected with 1 x 106 of NIH3T3-RETC634W cells and treatable tumors (~100 mm3) developed after 1 wk. Mice were treated with the indicated dose of drug or vehicle daily by gavage. Tumor growth and body weight were monitored twice weekly. Tumor volumes were determined according to the formula: length x diameter2 x {pi}/6. *, P < 0.05, statistically significant change in tumor volume versus controls (ANOVA). B, Western blot analysis for phosphorylated (tyrosine 509, p-tyrosine) and total RET protein in NIH3T3-RETC634W tumors harvested 6 h following the final treatment.

 
In our hands, TPC-1 cells did not grow as readily as xenografts. Hence, in vivo experiments to test the effects of NVP-AST487 were done exclusively on TT cells. The data in Fig. 4A show that oral administration of NVP-AST487 at 50 or 30 mg/kg/d decreased mean tumor volume as compared with control mice (P < 0.001 ANOVA). These data were replicated in two additional efficacy experiments. Treatment of athymic mice was not associated with significant body weight change of the animals at any of the concentrations tested.


Figure 4
View larger version (8K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Rapid inhibition of human plasma calcitonin levels by NVP-AST487 independent of effects on tumor volume in athymic mice with TT cell xenografts. A, female athymic nude mice were s.c. injected with 5 x 106 TT cells and treatable tumors (~100 mm3) developed after 3 weeks. Mice were treated with the indicated dose of drug or vehicle daily by gavage. *, P < 0.05, statistically significant change in tumor volume versus controls (ANOVA). B, female athymic nude mice bearing TT cell tumor xenografts were treated with a single dose of 50 mg/kg NVP-AST487 p.o., and plasma calcitonin and tumor weight measured 4 days later (n = 4; P = 0.01 when calcitonin concentrations were normalized for tumor weight).

 
A notable finding from these experiments was that the plasma levels of human calcitonin, which were measured as a biomarker of tumor responsiveness to NVP-AST487, seemed to decrease prior to any changes in tumor volume. As shown in Fig. 4B, plasma calcitonin levels measured 96 h after a single oral administration of 50 mg/kg of NVP-AST487 were markedly decreased, at a time when tumor volume was still unchanged. This difference was statistically significant when the calcitonin concentrations were normalized to the tumor weight (P < 0.05; t test), but not when absolute values were analyzed (P = 0.083).

NVP-AST487 directly inhibits calcitonin gene expression, independent of effects on TT cell growth. We next examined the effects of NVP-AST487 on calcitonin gene expression in vitro. As shown in Fig. 5A , treatment of TT cells with 100 nmol/L of NVP-AST487 was associated with an ~50% decrease in calcitonin mRNA after 96 h. The quantification of this effect in three independent biological replicates is shown as supplemental data. The delayed response to the compound is likely due to the prolonged half-life of calcitonin mRNA, as inhibition of nascent transcription by treatment with actinomycin D resulted in a very modest decay of mature calcitonin mRNA over a 24 h incubation (supplemental data), which is consistent with the previously reported t1/2 of calcitonin mRNA in TT cells (20). Calcitonin secretion into conditioned media was markedly inhibited by NVP-AST487 in a concentration-dependent fashion, with maximal effects observed at 50 to 100 nmol/L (Fig. 5B). Besides calcitonin, MTC cells also secrete CEA, which is also a valuable tumor marker. NVP-AST487 had no effect on CEA mRNA levels (supplemental data). To determine whether NVP-AST487 inhibited calcitonin gene transcription, we transiently transfected TT cells with the calcitonin promoter plasmid, pCT252CAT, the activity of which is induced by RAS in these cells (21). Treatment with 100 nmol/L of NVP-AST487 resulted in a 3-fold inhibition of promoter activity after 48 h. The pCT132 promoter fragment that lacks the domain conferring responsiveness to RAS also seemed to be inhibited by the kinase inhibitor (Fig. 5C). The effects of NVP-AST487 were unlikely to be due to off-target actions because PP2 and ZD6474, compounds previously shown to inhibit RET kinase activity (16, 22), also markedly decreased calcitonin mRNA levels in TT cells after 72 h (Fig. 5D). All three compounds inhibited CGRP mRNA to a comparable level. This is consistent with an effect on the abundance of the primary CTN/CGRP transcript rather than through regulation of alternative splicing of the gene.


Figure 5
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. NVP-AST487 decreases TT cell calcitonin gene transcription, mRNA abundance, and secretion in vitro. A, representative Northern blot of TT cell calcitonin mRNA at the indicated times after incubation with 100 nmol/L of NVP-AST487. Quantitation of effects of NVP-AST487 on calcitonin mRNA is presented in supplemental data. B, calcitonin concentration in conditioned media of TT cells incubated with the indicated concentration of NVP-AST487 for 96 h, with media exchange at 48 h. Columns, means of three independent experiments expressed relative to DNA content of cells harvested from the wells after removal of the conditioned media; bars, SE. C, treatment of TT cells with NVP-AST487 inhibits calcitonin gene transcription. TT cells were seeded onto six-well plates and transiently cotransfected with 2 µg of pCT132CAT or pCT252CAT and 0.4 µg of the pGL3-control. CAT protein and luciferase were quantified 48 h later. Columns, normalized CAT/luciferase levels in cells treated with or without NVP-AST487. Similar findings were obtained in three additional biological replicates. D, effects of 72-h incubation of TT cells with NVP-AST487 (100 nmol/L), PP2 (5 µmol/L), or ZD6474 (500 nmol/L) on CTN/CGRP mRNA measured by quantitative reverse transcription-PCR.

 
Persephin and GDNF induce calcitonin gene expression in MTC-M cells. As calcitonin gene expression is inhibited by the RET kinase antagonist NVP-AST487, we next examined whether the converse is also the case, i.e., whether ligand-induced RET activation induces calcitonin mRNA levels. To this end, we used the mouse medullary thyroid cancer cell line, MTC-M, which was shown to have a wild-type sequence (see Materials and Methods).

MTC-M cells were treated with GDNF in the presence or absence of recombinant rat GFR{alpha}1/Fc chimeric protein, which binds GDNF in solution and activates RET kinase (23). GDNF alone evoked only a faint induction of calcitonin mRNA (data not shown); however, in the presence of GFR{alpha}1, GDNF induced a marked increase in calcitonin mRNA levels (Fig. 6A ). By contrast to GDNF, treatment of cells with persephin alone induced calcitonin mRNA (Fig. 6B and C), which was only modestly augmented by coincubation with GFR{alpha}1 (data not shown). Both GDNF/GFR{alpha}1 and persephin-induced calcitonin mRNA were markedly inhibited by coincubation with 100 nmol/L of NVP-AST487 (Fig. 6B). The CTN/CGRP gene has been shown to be mitogen-activated protein kinase (MAPK)–responsive. Accordingly, the MEK inhibitor UO126 also inhibited persephin-induced calcitonin mRNA levels (Fig. 6C).


Figure 6
View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. GDNF (in the presence of GFR{alpha}-1) and persephin induce calcitonin mRNA in MTC-M cells. A, MTC-M cells were placed in serum-free media for 16 h and then treated with GDNF (200 ng/mL) and GFR{alpha}-1 (1 µg/mL) for the indicated times. Northern blots of RNA extracts were probed with a mouse calcitonin cDNA probe. B, NVP-AST487 (100 nmol/L) inhibits persephin (200 ng/mL) and GDNF + GFR{alpha}-1–induced calcitonin mRNA. Cells were treated as in (A) in the presence of NVP-AST487 or vehicle for 24 h, and calcitonin mRNA levels assessed by Northern blotting. C, the MEK inhibitor UO126 inhibits persephin-induced calcitonin mRNA. MTC-M cells were treated with or without persephin or UO126 (15 µmol/L) for 24 h. Top, representative Northern blot.

 
NVP-AST487 inhibits plasma calcitonin levels in mice. We next examined the effects of NVP-AST487 administered at 50 mg/kg/d by gavage on plasma murine calcitonin levels in FVB-N wild-type mice. As shown in Fig. 7 , plasma calcitonin levels decreased rapidly, and were significantly lower than controls by 8 h after administration of a single dose p.o. (Fig. 7A). Levels remained significantly lower than vehicle-treated controls up to 72 h (Fig. 7B). There was no corresponding decrease in thyroid tissue calcitonin mRNA at these time points (data not shown), suggesting that at least part of the inhibitory effects of NVP-AST487 in vivo are posttranscriptional.


Figure 7
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 7. NVP-AST487 inhibits plasma calcitonin levels in wild-type mice. A, 6- to 8-week-old FVBN mice were treated with a single dose of 50 mg/kg NVP-AST487 by oral gavage and plasma collected at 2 and 8 h after treatment (n = 5 per time point; *, P < 0.015 versus time 0). B, 6- to 8-week-old mice were treated with daily administration of 50 mg/kg/d p.o. NVP-AST487 or vehicle and plasma collected at the indicated times (n = 4 to 5 per time point for NVP-AST487–treated; n = 3–5 for vehicle-treated animals; *, P < 0.02 and **, P < 0.001 versus corresponding vehicle-treated controls).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Some of the most striking early successes of targeted cancer therapies with kinase inhibitors have been with compounds that block the activity of oncoproteins that are involved in the early stages of tumor development, and which are believed to be required for the continued viability of the neoplastic clone. BCR-ABL in chronic myelogenous leukemia and KIT in gastrointestinal stromal tumors meet these criteria, and likely account for the sensitivity of these cancers to imatinib, which effectively inhibits the activity of these kinases in human cancers. The fact that activating mutations of RET are present in the germ line of almost all patients with familial forms of MTC indicates that this oncogene is a very early event in the development of these cancers. Moreover, the requirement for continued activity of RET for MTC progression is suggested by evidence that orthotopic thyroidal injection of adenoviral vectors directing the expression of a dominant-negative RET kinase antagonist impairs the growth of MTC in a transgenic mouse line with C cell–specific expression of oncogenic RET (24). Familial MTCs may represent the cancer type in which the key event in tumor initiation is best established, and might thus be a good paradigm in which to test the hypothesis that cancers remain dependent on the continued unregulated activation of a tumor-initiating event for their growth or survival.

This potential has not escaped the attention of other investigators. Several groups have characterized compounds with inhibitory activity on RET kinase (reviewed in ref. 12). One of them in particular, the quinazoline ZD6474, was found to have an IC50 for GST-RET/TK of ~130 nmol/L. This compound inhibited the activity of most of the common oncogenic mutants of RET, including the RET/PTC fusion genes involved in the development of papillary thyroid cancers, with the notable exception of RETV804M. This particular substitution corresponds to residues in ABL, EGFR, and PDGFR, which have been shown to mediate resistance to various kinase inhibitors (25). ZD6474 is presently in phase 2 clinical trials for familial MTC, the results of which have not yet been published.

Here, we report the characterization of a novel compound that is structurally distinct from other RET kinase inhibitors. NVP-AST487 exhibits a potent growth-inhibitory effect in RET-dependent Baf3 cells, and in TT and TPC-1 cells, derived, respectively, from a MTC and PTC harboring activating RET mutations. By contrast, the compound had no effect on other thyroid cancer cell lines, most of which had activating mutations of BRAF. The selectivity of action of NVP-AST487 on thyroid cancer cell lines with RET mutations argues for the requirement of RET kinase activity for their continued viability.

There was a noticeable difference in the inhibitory activity of the compound in vitro, in which the IC50 was 0.88 µmol/L, as compared with cells in which the compound was at least 10-fold more potent. There are several potential explanations for this. First, the two assays of RET activity are distinct: in vitro, we assayed the ability of a GST-RET fusion protein to phosphorylate a substrate, whereas in cells we measured RET autophosphorylation by Western blotting. Concentrations of ATP, the binding competitor for NVP-AST487, may be quite different in these two settings, which could be critical. Moreover, NVP-AST487 is predicted to bind to RET in its inactive conformation, which is more likely to occur in whole cells than in the GST fusion protein in vitro. The inhibitory effects of the compound on other kinases, such as Abl, may also contribute to the greater potency of NVP-AST487 in blocking the growth of thyroid cancer cell lines expressing oncogenic RET, particularly as RET-induced Abl activation mediates phosphorylation of the MAPK protein Erk8 (26).

Calcitonin belongs to a family of structurally related but biologically distinct molecules that includes calcitonin itself, its splice variant CGRP, amylin, and adrenomedulin. The calcitonin gene family consists of four genes (CALC-I to CALC-IV). Calcitonin and its splice variant CGRP-1 are differentially spliced products of CALC-I in C cells and neuronal cells, respectively. Plasma calcium concentration is a primary physiologic stimulus of calcitonin secretion in thyroid parafollicular C cells, an effect that is mediated through the calcium-sensing receptor (27). Other than its regulation via calcium sensing, calcitonin gene expression in C cells also responds to peptide hormones. The gastrointestinal hormone gastrin induces rapid calcitonin secretion as well as gene transcription in TT cells, acting via the G protein–coupled cholecystokinin 2 receptor (CCK2R; ref. 28), which may explain the brisk calcitonin response to pentagastrin seen in patients with MTC. There are indications that CCK2R is also expressed in normal parafollicular C cells (29). Glucagon-like peptide I also induces calcitonin mRNA in rat MTC cells (30).

The inhibition of human calcitonin secretion from TT cell xenografts by NVP-AST487 in vivo is consistent with a direct effect of the compound on calcitonin gene expression, in all likelihood, through the inhibition of RET kinase activity. Several lines of evidence substantiate this interpretation of the data. NVP-AST487 inhibits calcitonin mRNA abundance in TT cells in vitro. The relatively slow kinetics of calcitonin mRNA decay may be due to the previously reported prolonged t1/2 of the transcript (20), which we confirmed. NVP-AST487 inhibited calcitonin gene transcription directed by a CT promoter fragment that contained an element previously reported to be activated via RAS (21). As RET activates RAS and MAPK, it is possible that this pathway may be a primary driver of calcitonin gene transcription. Clearly, other factors contribute significantly to calcitonin gene regulation, which can also be activated through cyclic AMP (31, 32). The evidence that RET kinase inhibition blocked calcitonin gene expression led us to explore the hypothesis that ligand-induced RET activation resulted in a reciprocal effect. To this end, it was critical to identify a C cell line that expressed wild-type RET because activated mutants of this tyrosine kinase receptor oncogene are either constitutively fully active or show attenuated responses to ligand. The mouse MTC-M cell line was found to express a wild-type RET gene product, and was thus suitable for these experiments. Treatment of these cells with GDNF alone elicited no response, consistent with the notion that GDNF may not be the natural ligand for RET in C cells, as they do not express GFR{alpha}1, the coreceptor for GDNF, but instead preferentially express GFR{alpha}4, which is the binding receptor for persephin (33). Indeed, costimulation of MTC-M cells with both GDNF and a soluble form of GFR{alpha}1 robustly induced calcitonin mRNA levels. By contrast, treatment with persephin alone was sufficient to stimulate calcitonin gene expression. RET kinase activity was required for ligand-induced calcitonin gene expression because it was blocked by NVP-AST487. Moreover, the contribution of MAPK activity to persephin-induced calcitonin gene expression was found to be significant because the effects were partially blocked by UO126.

While our study was being written, Lindfors et al. reported the phenotype of mice with ablation of the persephin coreceptor GFR{alpha}4 gene (34). These animals had a normal number of C cells with apparently preserved morphology, but had decreased calcitonin content in the thyroid of neonates and young animals. Although calcitonin content in the thyroid returned to normal in adult animals, these data point to a physiologic requirement for signaling via GFR{alpha}4-RET for calcitonin gene expression in C cells. Further evidence that this novel calcitonin regulatory pathway is physiologic is provided by our observation that NVP-AST487 markedly reduced plasma calcitonin levels in normal mice.

Oral administration of NVP-AST487 was associated with a significant decline in plasma calcitonin levels at 8 h. A similar early drop in plasma human calcitonin was seen in athymic mice with TT cell xenografted tumors (data not shown). In view of the prolonged t1/2 of calcitonin mRNA, this suggests that RET signaling may be mediating distinct effects on calcitonin secretion and gene expression. Although the former mechanism remains to be proven, a dual mechanism of regulation of calcitonin gene expression and secretion by gastrin has also been proposed (28).

The findings reported here are of potential clinical significance because plasma calcitonin is used routinely as an indicator of progression in patients with persistent or recurrent MTC, as it is believed to be roughly proportional to tumor mass. Now that small-molecule RET kinase inhibitors are being evaluated for therapeutic effectiveness in patients with metastatic MTC, our data raises the possibility that measurement of calcitonin may not accurately reflect tumor burden in these patients. On the other hand, evidence of a rapid decrease in calcitonin levels after short-term treatment with a RET kinase inhibitor may provide indirect evidence that the kinase has been effectively targeted. In a larger context, these data point to the need to reappraise the performance of tumor biomarkers when new therapies are being tested, particularly when they are directed against cellular targets believed to be of pathogenetic significance.


   Acknowledgments
 
Grant support: NIH grants CA72597, CA50706, and a grant from Novartis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org).

Received 12/14/06. Revised 3/21/07. Accepted 5/11/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Airaksinen MS, Saarma M. The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 2002;3:383–94.[CrossRef][Medline]
  2. Baloh RH, Enomoto H, Johnson EM, Jr., Milbrandt J. The GDNF family ligands and receptors—implications for neural development. Curr Opin Neurobiol 2000;10:103–10.[CrossRef][Medline]
  3. Avantaggiato V, Dathan NA, Grieco M, et al. Developmental expression of the RET protooncogene. Cell Growth Differ 1994;5:305–11.[Abstract]
  4. Pachnis V, Mankoo B, Costantini F. Expression of the c-ret proto-oncogene during mouse embryogenesis. Development 1993;119:1005–17.[Abstract]
  5. Nakamura T, Ishizaka Y, Nagao M, Hara M, Ishikawa T. Expression of the ret proto-oncogene product in human normal and neoplastic tissues of neural crest origin. J Pathol 1994;172:255–60.[CrossRef][Medline]
  6. Santoro M, Melillo RM, Carlomagno F, Fusco A, Vecchio G. Molecular mechanisms of RET activation in human cancer. Ann N Y Acad Sci 2002;963:116–21.[Medline]
  7. Schuchardt A, D'Agati V, Larsson-Blomberg L, Costantini F, Pachnis V. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret [see comments]. Nature 1994;367:380–3.[CrossRef][Medline]
  8. Enomoto H, Crawford PA, Gorodinsky A, Heuckeroth RO, Johnson EM, Jr., Milbrandt J. RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 2001;128:3963–74.[Medline]
  9. Lee S, Nakamura E, Yang H, Wei W, Linggi MS, Sajan MP, et al. Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer. Cancer Cell 2005;8:155–67.[CrossRef][Medline]
  10. Santoro M, Melillo RM, Grieco M, Berlingieri MT, Vecchio G, Fusco A. The TRK and RET tyrosine kinase oncogenes cooperate with ras in the neoplastic transformation of a rat thyroid epithelial cell line. Cell Growth Differ 1993;4:77–84.[Abstract]
  11. Ishizaka Y, Takahashi M, Ushijima T, Sugimura T, Nagao M. A high phosphorylation state and increased activity of the TRE motif in the NIH3T3 cell transformant induced by retTPC. Biochem Biophys Res Commun 1991;179:1331–6.[CrossRef][Medline]
  12. Carlomagno F, Santoro M. Receptor tyrosine kinases as targets for anticancer therapeutics. Curr Med Chem 2005;12:1773–81.[CrossRef][Medline]
  13. Carlomagno F, Vitagliano D, Guida T, et al. The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes. Cancer Res 2002;62:1077–82.[Abstract/Free Full Text]
  14. Strock CJ, Park JI, Rosen M, et al. CEP-701 and CEP-751 inhibit constitutively activated RET tyrosine kinase activity and block medullary thyroid carcinoma cell growth. Cancer Res 2003;63:5559–63.[Abstract/Free Full Text]
  15. Lanzi C, Cassinelli G, Cuccuru G, et al. Inactivation of Ret/Ptc1 oncoprotein and inhibition of papillary thyroid carcinoma cell proliferation by indolinone RPI-1. Cell Mol Life Sci 2003;60:1449–59.[CrossRef][Medline]
  16. Carlomagno F, Vitagliano D, Guida T, et al. ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Res 2002;62:7284–90.[Abstract/Free Full Text]
  17. Ishizaka Y, Ushijima T, Sugimura T, Nagao M. cDNA cloning and characterization of ret activated in a human papillary thyroid carcinoma cell line. Biochem Biophys Res Commun 1990;168:402–8.[CrossRef][Medline]
  18. Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 2003;63:1454–7.[Abstract/Free Full Text]
  19. Nikiforova MN, Kimura ET, Gandhi M, et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab 2003;88:5399–404.[Abstract/Free Full Text]
  20. Nelkin BD, Chen KY, De Bustros A, Roos BA, Baylin SB. Changes in calcitonin gene RNA processing during growth of a human medullary thyroid carcinoma cell line. Cancer Res 1989;49:6949–52.[Abstract/Free Full Text]
  21. Thiagalingam A, De Bustros A, Borges M, et al. RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas. Mol Cell Biol 1996;16:5335–45.[Abstract]
  22. Carlomagno F, Vitagliano D, Guida T, Basolo F, Castellone MD, Melillo RM, et al. Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). J Clin Endocrinol Metab 2003;88:1897–902.[Abstract/Free Full Text]
  23. Treanor JJ, Goodman L, de Sauvage F, et al. Characterization of a multicomponent receptor for GDNF. Nature 1996;382:80–3.[CrossRef][Medline]
  24. Drosten M, Hilken G, Bockmann M, et al. Role of MEN2A-derived RET in maintenance and proliferation of medullary thyroid carcinoma. J Natl Cancer Inst 2004;96:1231–9.[Abstract/Free Full Text]
  25. Carlomagno F, Guida T, Anaganti S, et al. Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors. Oncogene 2004;23:6056–63.[CrossRef][Medline]
  26. Iavarone C, Acunzo M, Carlomagno F, et al. Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene. J Biol Chem 2006;281:10567–76.[Abstract/Free Full Text]
  27. Fudge NJ, Kovacs CS. Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release in vivo. BMC Physiol 2004;4:5.[CrossRef][Medline]
  28. Blaker M, Arrenberg P, Stange I, et al. The cholecystokinin2-receptor mediates calcitonin secretion, gene expression, and proliferation in the human medullary thyroid carcinoma cell line, TT. Regul Pept 2004;118:111–7.[CrossRef][Medline]
  29. Blaker M, de Weerth A, Tometten M, et al. Expression of the cholecystokinin 2-receptor in normal human thyroid gland and medullary thyroid carcinoma. Eur J Endocrinol 2002;146:89–96.[Abstract]
  30. Lamari Y, Boissard C, Moukhtar MS, Jullienne A, Rosselin G, Garel JM. Expression of glucagon-like peptide 1 receptor in a murine C cell line: regulation of calcitonin gene by glucagon-like peptide 1. FEBS Lett 1996;393:248–52.[CrossRef][Medline]
  31. De Bustros A, Lee RY, Compton D, Tsong TY, Baylin SB, Nelkin BD. Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma. Mol Cell Biol 1990;10:1773–8.[Abstract/Free Full Text]
  32. De Bustros A, Ball DW, Peters R, Compton D, Nelkin BD. Regulation of human calcitonin gene transcription by cyclic AMP. Biochem Biophys Res Commun 1992;189:1157–64.[CrossRef][Medline]
  33. Masure S, Cik M, Hoefnagel E, et al. Mammalian GFR{alpha}-4, a divergent member of the GFR{alpha} family of coreceptors for glial cell line-derived neurotrophic factor family ligands, is a receptor for the neurotrophic factor persephin. J Biol Chem 2000;275:39427–34.[Abstract/Free Full Text]
  34. Lindfors PH, Lindahl M, Rossi J, Saarma M, Airaksinen MS. Ablation of persephin receptor glial cell line-derived neurotrophic factor family receptor {alpha}4 impairs thyroid calcitonin production in young mice. Endocrinology 2006;147:2237–44.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
S. I. Sherman
Advances in Chemotherapy of Differentiated Epithelial and Medullary Thyroid Cancers
J. Clin. Endocrinol. Metab., May 1, 2009; 94(5): 1493 - 1499.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. Weisberg, J. Roesel, G. Bold, P. Furet, J. Jiang, J. Cools, R. D. Wright, E. Nelson, R. Barrett, A. Ray, et al.
Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells
Blood, December 15, 2008; 112(13): 5161 - 5170.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
S. V. Sharma and J. Settleman
Oncogene addiction: setting the stage for molecularly targeted cancer therapy
Genes & Dev., December 15, 2007; 21(24): 3214 - 3231.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. Plaza-Menacho, L. Mologni, E. Sala, C. Gambacorti-Passerini, A. I. Magee, T. P. Links, R. M. W. Hofstra, D. Barford, and C. M. Isacke
Sorafenib Functions to Potently Suppress RET Tyrosine Kinase Activity by Direct Enzymatic Inhibition and Promoting RET Lysosomal Degradation Independent of Proteasomal Targeting
J. Biol. Chem., October 5, 2007; 282(40): 29230 - 29240.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Akeno-Stuart, N.
Right arrow Articles by Fagin, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Akeno-Stuart, N.
Right arrow Articles by Fagin, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online